Antibodies to MAdCAM

ABSTRACT

The present invention relates to antibodies including human antibodies and antigen-binding portions thereof that specifically bind to MAdCAM, preferably human MAdCAM and that function to inhibit MAdCAM. The invention also relates to human anti-MAdCAM antibodies and antigen-binding portions thereof. The invention also relates to antibodies that are chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulins derived from human anti-MAdCAM antibodies and nucleic acid molecules encoding such immunoglobulins. The present invention also relates to methods of making human anti-MAdCAM antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-MAdCAM antibodies. The invention also relates to transgenic animals or plants comprising nucleic acid molecules of the invention.

This application is a divisional application of U.S. patent applicationSer. No. 15/086,930, now U.S. Pat. No. 10,259,872, issued Apr. 16, 2019,which is a divisional application of U.S. patent application Ser. No.11/031,485, filed Jan. 7, 2005, now U.S. Pat. No. 9,328,169, issued May3, 2016, which claims the benefit of U.S. provisional Application60/535,490, filed Jan. 9, 2004 (expired), which applications areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 000659-0055-101-SL. The text file is 191,159bytes in size, was created on Mar. 31, 2016, and is being submittedelectronically via EFS Web.

BACKGROUND OF THE INVENTION

Mucosal addressin cell adhesion molecule (MAdCAM) is a member of theimmunoglobulin superfamily of cell adhesion receptors. The selectivityof lymphocyte homing to specialized lymphoid tissue and mucosal sites ofthe gastrointestinal tract is determined by the endothelial expressionof MAdCAM (Berlin, C. et al., Cell, 80:413-422 (1994); Berlin, C., etal., Cell, 74:185-195 (1993); and Erle, D. J., et al., J. Immunol., 153:517-528 (1994)). MAdCAM is uniquely expressed on the cell surface ofhigh endothelial venules of organized intestinal lymphoid tissue, suchas Peyer's patches and mesenteric lymph nodes (Streeter et al., Nature,331:41-6 (1988); Nakache et al., Nature, 337:179-81 (1989); Briskin etal., Am. J. Pathol. 151-97-110 (1997)), but also in other lymphoidorgans, such as pancreas, gall bladder and splenic venules and marginalsinus of the splenic white pulp (Briskin et al (1997), supra; Kraal etal., Am. J. Path., 147: 763-771 (1995)).

While MAdCAM plays a physiological role in gut immune surveillance, itappears to facilitate excessive lymphocyte extravasation in inflammatorybowel disease under conditions of chronic gastrointestinal tractinflammation. TNFα and other pro-inflammatory cytokines increaseendothelial MAdCAM expression and, in biopsy specimens taken frompatients with Crohn's disease and ulcerative colitis, there is anapproximate 2-3 fold focal increase in MAdCAM expression at sites ofinflammation (Briskin et al. (1997), Souza et al., Gut, 45:856-63(1999); Arihiro et al., Pathol Int., 52:367-74 (2002)). Similar patternsof elevated expression have been observed in experimental models ofcolitis (Hesterberg et al., Gastroenterology, 111:1373-1380 (1997);Picarella et al., J. Immunol., 158: 2099-2106 (1997); Connor et al., JLeukoc Biol., 65:349-55 (1999); Kato et al., J Pharmacol Exp Ther.,295:183-9 (2000); Hokari et al., Clin Exp Immunol., 26:259-65 (2001);Shigematsu et al., Am J Physiol Gastrointest Liver Physiol.,281:G1309-15 (2001)). In other pre-clinical models for inflammatoryconditions, such as insulin-dependent diabetes (Yang et al. Diabetes,46:1542-7 (1997); Hänninen et al., J Immunol., 160:6018-25 (1998)),graft versus host disease (Fujisaki et al., Scand J Gastroenterol.,38:437-42 (2003), Murai et al., Nat Immunol., 4:154-60 (2003)), chronicliver disease (Hillan et al., Liver, 19:509-18 (1999); Grant et al.,Hepatology, 33:1065-72 (2001)), inflammatory encephalopathy (Stalder etal., Am J Pathol., 153:767-83 (1998); Kanawar et al., Immunol CellBiol., 78:641-5 (2000)), and gastritis (Barrett et al., J Leukoc Biol.,67:169-73 (2000); Hatanaka et al., Clin Exp Immunol., 130:183-9 (2002)),there is also reawakening of fetal MAdCAM expression and participationof activated α₄β₇ ⁺ lymphocytes in disease pathogenesis. In theseinflammatory models as well as hapten-mediated (e.g., TNBS, DSS, etc.)or adoptive transfer (CD4⁺CD45Rb^(high)) mouse colitic models, the ratanti-mouse MAdCAM monoclonal antibody (mAb), MECA-367, which blocks thebinding of α₄β₇ ⁺ lymphocytes to MAdCAM, reduces the lymphocyterecruitment, tissue extravasation, inflammation and disease severity.Mouse monoclonal antibodies (mAbs) against human MAdCAM also have beenreported (see, e.g., WO 96/24673 and WO 99/58573).

Given the role of MAdCAM in inflammatory bowel disease (IBD) and otherinflammatory diseases associated with the gastrointestinal tract orother tissues, a means for inhibiting α₄β₇ ⁺ binding and MAdCAM-mediatedleukocyte recruitment is desirable. It further would be desirable tohave such therapeutic means with advantageous properties including butnot limited to the absence of unwanted interactions with othermedications in patients and favorable physico-chemical properties suchas pK/pD values in humans, solubility, stability, shelf-life and in vivohalf-life. A therapeutic protein, such as an antibody, wouldadvantageously be free of unwanted post-translational modifications oraggregate formation. Accordingly, there is a critical need fortherapeutic anti-MAdCAM antibodies.

SUMMARY OF THE INVENTION

The present invention provides an isolated antibody that specificallybinds MAdCAM, wherein at least the CDR sequences of said antibody arehuman CDR sequences, or an antigen-binding portion of said antibody. Insome embodiments the antibody is a human antibody, preferably anantibody that acts as a MAdCAM antagonist. Also provided arecompositions comprising said antibodies or portions.

The invention also provides a composition comprising the heavy and/orlight chain of said anti-MAdCAM antagonist antibody or the variableregion or other antigen-binding portion thereof or nucleic acidmolecules encoding any of the foregoing and a pharmaceuticallyacceptable carrier. Compositions of the invention may further compriseanother component, such as a therapeutic agent or a diagnostic agent.Diagnostic and therapeutic methods are also provided by the invention.

The invention further provides an isolated cell line, that produces saidanti-MAdCAM antibody or antigen-binding portion thereof.

The invention also provides nucleic acid molecules encoding the heavyand/or light chain of said anti-MAdCAM antibody or the variable regionthereof or antigen-binding portion thereof.

The invention provides vectors and host cells comprising said nucleicacid molecules, as well as methods of recombinantly producing thepolypeptides encoded by the nucleic acid molecules.

Non-human transgenic animals or plants that express the heavy and/orlight chain of said anti-MAdCAM antibody, or antigen-binding portionthereof, are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1T are alignments of the predicted amino acid sequences of theheavy and kappa light chain variable regions of twelve human anti-MAdCAMmonoclonal antibodies with the germline amino acid sequences of thecorresponding human genes.

FIG. 1A shows an alignment of the predicted amino acid sequence of theheavy chain for antibodies 1.7.2 and 1.8.2 with the germline human VH3-15 gene product.

FIG. 1B shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 6.14.2 with the germline human VH 3-23 geneproduct.

FIG. 1C shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 6.22.2 with the germline human VH 3-33 geneproduct.

FIG. 1D shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 6.34.2 with the germline human VH 3-30 geneproduct

FIG. 1E shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 6.67.1 with the germline human VH 4-4 geneproduct.

FIG. 1F shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 6.73.2 with the germline human VH 3-23 geneproduct.

FIG. 1G shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 6.77.1 with the germline human VH 3-21 geneproduct.

FIG. 1H shows an alignment of the predicted amino acid sequence of theheavy chain for antibodies 7.16.6 and 7.26.4 with the germline human VH1-18 gene product.

FIG. 1I shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 7.20.5 with the germline human VH 4-4 geneproduct.

FIG. 1J shows an alignment of the predicted amino acid sequence of theheavy chain for antibody 9.8.2 with the germline human VH 3-33 geneproduct.

FIG. 1K shows an alignment of the predicted amino acid sequence of thelight kappa chain for antibodies 1.7.2 and 1.8.2 with the germline humanA3 gene product.

FIG. 1L shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 6.14.2 with the germline human O12 geneproduct.

FIG. 1M shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 6.22.2 with the germline human A26 geneproduct.

FIG. 1N shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 6.34.2 with the germline human O12 geneproduct.

FIG. 1O shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 6.67.1 with the germline human B3 geneproduct.

FIG. 1P shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 6.73.2 with the germline human O12 geneproduct.

FIG. 1Q shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 6.77.1 with the germline human A2 geneproduct.

FIG. 1R shows an alignment of the predicted amino acid sequence of thekappa light chain for antibodies 7.16.6 and 7.26.4 with the germlinehuman A2 gene product.

FIG. 1S shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 7.20.5 with the germline human A3 geneproduct.

FIG. 1T shows an alignment of the predicted amino acid sequence of thekappa light chain for antibody 9.8.2 with the germline human 018 geneproduct.

FIGS. 2A and 2B are CLUSTAL alignments of the predicted heavy and kappalight chain amino acid sequences of human anti-MAdCAM antibodies.

FIG. 2A is a CLUSTAL alignment and radial tree of the predicted kappalight chain amino acid sequences, showing the degree of similaritybetween the anti-MAdCAM antibody kappa light chains.

FIG. 2B is a CLUSTAL alignment and radial tree of the predicted heavyamino acid sequences, showing the degree of similarity between theanti-MAdCAM antibody heavy chains.

FIG. 3 is an amino acid sequence CLUSTAL alignment of the 2 N-terminaldomains of cynomolgus and human MAdCAM which form the α₄β₇ bindingdomain. The β-strands are aligned according to Tan et al., Structure(1998) 6:793-801.

FIG. 4 is a graph representing the dose effects of purified biotinylated1.7.2 and 7.16.6 on the adhesion of human peripheral blood lymphocytesto sections of MAdCAM-expressing frozen human liver endothelium.

FIG. 5 shows a two dimensional graphical representation based on thedata captured in Table 7 of the diversity of MAdCAM epitopes to whichthe anti-MAdCAM antibodies, 1.7.2, 6.22.2, 6.34.2, 6.67.1, 6.77.1,7.16.6, 7.20.5, 7.26.4, 9.8.2 bind. Anti-MAdCAM antibodies within thesame circle show the same reactivity pattern, belong in the same epitopebin and are likely to recognize the same epitope on MAdCAM. Anti-MAdCAMantibody clones within overlapping circles are unable to bindsimultaneously and are, therefore, likely to recognize an overlappingepitope on MAdCAM. Non-integrating circles represent anti-MAdCAMantibody clones with distinct spatial epitope separation.

FIG. 6 shows sandwich ELISA data with anti-MAdCAM antibodies 1.7.2 andan Alexa 488-labelled 7.16.6, showing that two antibodies that are ableto detect different epitopes on MAdCAM could be used to detect solubleMAdCAM for diagnostic purposes.

FIG. 7 shows the effect of an inhibitory anti-MAdCAM antibody (1 mg/kg)on the number of circulating peripheral α₄β₇ ⁺ lymphocytes, expressed asa fold increase over control IgG₂a mAb or vehicle, using anti-MAdCAM mAb7.16.6 in a cynomolgus monkey model.

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, genetics,protein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art. The methods andtechniques of the present invention are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992), and Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) is free of other proteins from the same species(3) is expressed by a cell from a different species, or (4) does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and preferably will be over99% pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. In some embodiments,fragments are at least 5, 6, 8 or 10 amino acids long. In otherembodiments, the fragments are at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, even more preferably at least 70, 80, 90, 100, 150 or 200 aminoacids long.

The term “polypeptide analog” as used herein refers to a polypeptidethat comprises a segment of at least 25 amino acids that has substantialidentity to a portion of an amino acid sequence and that has at leastone of the following properties: (1) specific binding to MAdCAM undersuitable binding conditions, (2) ability to inhibit α₄β₇ ⁺ integrinand/or L-selectin binding to MAdCAM, or (3) ability to reduce MAdCAMcell surface expression in vitro or in vivo. Typically, polypeptideanalogs comprise a conservative amino acid substitution (or insertion ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, preferably at least 50, 60,70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often beas long as a full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, or (5) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al., Nature, 354:105 (1991), which are each incorporatedherein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics”. Fauchere, J. Adv. Drug Res., 15:29 (1986); Veber andFreidinger, TINS, p. 392 (1985); and Evans et al., J. Med. Chem.,30:1229 (1987), which are incorporated herein by reference. Suchcompounds are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a desired biochemical property or pharmacological activity), such asa human antibody, but have one or more peptide linkages optionallyreplaced by a linkage such as: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cisand trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods well known inthe art. Systematic substitution of one or more amino acids of aconsensus sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) may also be used to generate more stable peptides.In addition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387(1992), incorporated herein by reference); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

An “immunoglobulin” is a tetrameric molecule. In a naturally-occurringimmunoglobulin, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as κ and λlight chains. Heavy chains are classified as μ, δ, γ, α, or ϵ, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 or more aminoacids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)) (incorporated by reference in its entiretyfor all purposes). The variable regions of each light/heavy chain pairform the antibody binding site such that an intact immunoglobulin hastwo binding sites.

Immunoglobulin chains exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions to forman epitope-specific binding site. From N-terminus to C-terminus, bothlight and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3,CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987);Chothia et al., Nature, 342:878-883 (1989), each of which isincorporated herein by reference in their entirety.

An “antibody” refers to an intact immunoglobulin or to anantigen-binding portion thereof that competes with the intact antibodyfor specific binding. In some embodiments, an antibody is anantigen-binding portion thereof. Antigen-binding portions may beproduced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies. Antigen-binding portions include, interalia, Fab, Fab′, F(ab′)₂, Fv, dAb, and complementarity determiningregion (CDR) fragments, single-chain antibodies (scFv), chimericantibodies, diabodies and polypeptides that contain at least a portionof an immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide. A Fab fragment is a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; a F(ab)₂ fragment is abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; a Fd fragment consists of the VH and CH1domains; an Fv fragment consists of the VL and VH domains of a singlearm of an antibody; and a dAb fragment (Ward et al., Nature, 341:544-546(1989)) consists of a VH domain.

As used herein, an antibody that is referred to as, e.g., 1.7.2, 1.8.2,6.14.2, 6.34.2, 6.67.1, 6.77.2, 7.16.6, 7.20.5, 7.26.4 or 9.8.2, is amonoclonal antibody that is produced by the hybridoma of the same nameFor example, antibody 1.7.2 is produced by hybridoma 1.7.2. An antibodythat is referred to as 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or7.26.4-mod is a monoclonal antibody whose sequence has been modifiedfrom its corresponding parent by site-directed mutagenesis.

A single-chain antibody (scFv) is an antibody in which VL and VH regionsare paired to form a monovalent molecule via a synthetic linker thatenables them to be made as a single protein chain (Bird et al., Science,242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA,85:5879-5883 (1988)). Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) and Poljak, R. J., et al., Structure, 2:1121-1123(1994)). One or more CDRs from an antibody of the invention may beincorporated into a molecule either covalently or noncovalently to makeit an immunoadhesin that specifically binds to MAdCAM. An immunoadhesinmay incorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesinto specifically bind to a particular antigen of interest.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a “bispecific” or “bifunctional” antibody(diabody) has two different binding sites.

An “isolated antibody” is an antibody that (1) is not associated withnaturally-associated components, including other naturally-associatedantibodies, that accompany it in its native state, (2) is free of otherproteins from the same species, (3) is expressed by a cell from adifferent species, or (4) does not occur in nature. Examples of isolatedantibodies include an anti-MAdCAM antibody that has been affinitypurified using MAdCAM, an anti-MAdCAM antibody that has been produced bya hybridoma or other cell line in vitro, and a human anti-MAdCAMantibody derived from a transgenic mammal or plant.

As used herein, the term “human antibody” means an antibody in which thevariable and constant region sequences are human sequences. The termencompasses antibodies with sequences derived from human genes, butwhich have been changed, e.g., to decrease possible immunogenicity,increase affinity, eliminate cysteines or glycosylation sites that mightcause undesirable folding, etc. The term encompasses such antibodiesproduced recombinantly in non-human cells which might impartglycosylation not typical of human cells. The term also emcompassesantibodies which have been raised in a transgenic mouse which comprisessome or all of the human immunoglobulin heavy and light chain loci.

In one aspect, the invention provides a humanized antibody. In someembodiments, the humanized antibody is an antibody that is derived froma non-human species, in which certain amino acids in the framework andconstant domains of the heavy and light chains have been mutated so asto avoid or abrogate an immune response in humans. In some embodiments,a humanized antibody may be produced by fusing the constant domains froma human antibody to the variable domains of a non-human species.Examples of how to make humanized antibodies may be found in U.S. Pat.Nos. 6,054,297, 5,886,152 and 5,877,293. In some embodiments, ahumanized anti-MAdCAM antibody of the invention comprises the amino acidsequence of one or more framework regions of one or more humananti-MAdCAM antibodies of the invention.

In another aspect, the invention includes a “chimeric antibody”. In someembodiments the chimeric antibody refers to an antibody that containsone or more regions from one antibody and one or more regions from oneor more other antibodies. In a preferred embodiment, one or more of theCDRs are derived from a human anti-MAdCAM antibody of the invention. Ina more preferred embodiment, all of the CDRs are derived from a humananti-MAdCAM antibody of the invention. In another preferred embodiment,the CDRs from more than one human anti-MAdCAM antibody of the inventionare mixed and matched in a chimeric antibody. For instance, a chimericantibody may comprise a CDR1 from the light chain of a first humananti-MAdCAM antibody may be combined with CDR2 and CDR3 from the lightchain of a second human anti-MAdCAM antibody, and the CDRs from theheavy chain may be derived from a third anti-MAdCAM antibody. Further,the framework regions may be derived from one of the same anti-MAdCAMantibodies, from one or more different antibodies, such as a humanantibody, or from a humanized antibody.

A “neutralizing antibody,” “an inhibitory antibody” or antagonistantibody is an antibody that inhibits the binding of α₄β₇ ⁺ or α₄β₇⁺-expressing cells, or any other cognate ligand or cognateligand-expressing cells, to MAdCAM by at least about 20%. In a preferredembodiment, the antibody reduces inhibits the binding of α₄β₇ ⁺ integrinor α₄β₇ ⁺-expressing cells to MAdCAM by at least 40%, more preferably by60%, even more preferably by 80%, 85%, 90%, 95% or 100%. The bindingreduction may be measured by any means known to one of ordinary skill inthe art, for example, as measured in an in vitro competitive bindingassay. An example of measuring the reduction in binding of α₄β₇⁺-expressing cells to MAdCAM is presented in Example I.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specification.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known (Bowie etal., Science, 253:164 (1991)).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al., Ann. Biol. Clin.,51:19-26 (1993); Jonsson, U., et al., Biotechniques, 11:620-627 (1991);Johnsson, B., et al., J. Mol. Recognit., 8:125-131 (1995); and Johnnson,B., et al., Anal. Biochem., 198:268-277 (1991).

The term “k_(off)” refers to the off rate constant for dissociation ofan antibody from the antibody/antigen complex.

The term “K_(d)” refers to the dissociation constant of a particularantibody-antigen interaction. An antibody is said to bind an antigenwhen the dissociation constant is ≤1 μM, preferably ≤100 nM and mostpreferably ≤10 nM.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids orcarbohydrate side chains and usually have specific three dimensionalstructural characteristics, as well as specific charge characteristics.An epitope may be “linear” or “conformational.” In a linear epitope, allof the points of interaction between the protein and the interactingmolecule (such as an antibody) occur linearally along the primary aminoacid sequence of the protein. In a conformational epitope, the points ofinteraction occur across amino acid residues on the protein that areseparated from one another.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, 0-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes; although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention can be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res., 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, Chemical Reviews, 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein refersto polynucleotide sequences which are necessary to effect the expressionand processing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. “High stringency” or “highly stringent” conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. An example of “high stringency” or “highlystringent” conditions is a method of incubating a polynucleotide withanother polynucleotide, wherein one polynucleotide may be affixed to asolid surface such as a membrane, in a hybridization buffer of 6×SSPE orSSC, 50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA at a hybridization temperature of 42° C. for12-16 hours, followed by twice washing at 55° C. using a wash buffer of1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “percent sequence identity” in the context of nucleotidesequences refers to the residues in two sequences which are the samewhen aligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.3, Accelrys, SanDiego, Calif. FASTA, which includes, e.g., the programs FASTA2 andFASTA3, provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,Methods Enzymol., 183: 63-98 (1990); Pearson, Methods Mol. Biol., 132:185-219 (2000); Pearson, Methods Enzymol., 266: 227-258 (1996); Pearson,J. Mol. Biol., 276: 71-84 (1998); herein incorporated by reference).Unless otherwise specified, default parameters for a particular programor algorithm are used. For instance, percent sequence identity betweennucleotide sequences can be determined using FASTA with its defaultparameters (a word size of 6 and the NOPAM factor for the scoringmatrix) or using Gap with its default parameters as provided inWisconsin Package Version 10.3, herein incorporated by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid molecule havinga particular sequence should be understood to encompass itscomplementary strand, with its complementary sequence.

In the molecular biology art, researchers use the terms “percentsequence identity”, “percent sequence similarity” and “percent sequencehomology” interchangeably. In this application, these terms shall havethe same meaning with respect to nucleotide sequences only.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, indicates that,when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), thereis nucleotide sequence identity in at least about 85%, preferably atleast about 90%, and more preferably at least about 95%, 96%, 97%, 98%or 99% of the nucleotide bases, as measured by any well-known algorithmof sequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 75% or 80%sequence identity, preferably at least 90% or 95% sequence identity,even more preferably at least 98% or 99% sequence identity. Preferably,residue positions that are not identical differ by conservative aminoacid substitutions. A “conservative amino acid substitution” is one inwhich an amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent sequence identityor degree of similarity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. See, e.g.,Pearson, Methods Mol. Biol., 24: 307-31 (1994), herein incorporated byreference. Examples of groups of amino acids that have side chains withsimilar chemical properties include 1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl sidechains: serine and threonine; 3) amide-containing side chains:asparagine and glutamine; 4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, andhistidine; and 6) sulfur-containing side chains are cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science, 256: 1443-45 (1992), herein incorporated by reference.A “moderately conservative” replacement is any change having anonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG contains programs such as“Gap” and “Bestfit” which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., Wisconsin package Version 10.3. Polypeptide sequences also can becompared using FASTA using default or recommended parameters, a programin Wisconsin package Version 10.3. FASTA (e.g., FASTA2 and FASTA3)provides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson (1990);Pearson (2000)). Another preferred algorithm when comparing a sequenceof the invention to a database containing a large number of sequencesfrom different organisms is the computer program BLAST, especiallyblastp or tblastn, using default parameters. See, e.g., Altschul et al.,J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-402 (1997); herein incorporated by reference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.

As used herein, the terms “label” or “labeled” refers to incorporationof another molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g., a drug conjugate or toxin. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials. The term “pharmaceutical agent or drug” asused herein refers to a chemical compound or composition capable ofinducing a desired therapeutic effect when properly administered to apatient. Other chemistry terms herein are used according to conventionalusage in the art, as exemplified by The McGraw-Hill Dictionary ofChemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)),incorporated herein by reference).

The term “anti-inflammatory” or “immuno-modulatory” agent is used hereinto refer to agents that have the functional property of inhibitinginflammation, including inflammatory disease in a subject, including ina human. In various embodiments of this invention, the inflammatorydisease may be, but is not limited to inflammatory diseases of thegastrointestinal tract including Crohn's disease, ulcerative colitis,diverticula disease, gastritis, liver disease, primary biliarysclerosis, sclerosing cholangitis. Inflammatory diseases also includebut are not limited to abdominal disease (including peritonitis,appendicitis, biliary tract disease), acute transverse myelitis,allergic dermatitis (including allergic skin, allergic eczema, skinatopy, atopic eczema, atopic dermatitis, cutaneous inflammation,inflammatory eczema, inflammatory dermatitis, flea skin, miliarydermatitis, miliary eczema, house dust mite skin), ankylosingspondylitis (Reiters syndrome), asthma, airway inflammation,atherosclerosis, arteriosclerosis, biliary atresia, bladderinflammation, breast cancer, cardiovascular inflammation (includingvasculitis, rheumatoid nail-fold infarcts, leg ulcers, polymyositis,chronic vascular inflammation, pericarditis, chronic obstructivepulmonary disease), chronic pancreatitis, perineural inflammation,colitis (including amoebic colitis, infective colitis, bacterialcolitis, Crohn's colitis, ischemic colitis, ulcerative colitis,idiopathic proctocolitis, inflammatory bowel disease, pseudomembranouscolitis), collagen vascular disorders (rheumatoid arthritis, SLE,progressive systemic sclerosis, mixed connective tissue disease,diabetes mellitus), Crohn's disease (regional enteritis, granulomatousileitis, ileocolitis, digestive system inflammation), demyelinatingdisease (including myelitis, multiple sclerosis, disseminated sclerosis,acute disseminated encephalomyelitis, perivenous demyelination, vitaminB12 deficiency, Guillain-Barre syndrome, MS-associated retrovirus),dermatomyositis, diverticulitis, exudative diarrhea, gastritis,granulomatous hepatitis, granulomatous inflammation, cholecystitis,insulin-dependent diabetes mellitus, liver inflammatory diseases (liverfibrosis primary biliary cirrhosis, hepatitis, sclerosing cholangitis),lung inflammation (idiopathic pulmonary fibrosis, eosinophilic granulomaof the lung, pulmonary histiocytosis X, peribronchiolar inflammation,acute bronchitis), lymphogranuloma venereum, malignant melanoma,mouth/tooth disease (including gingivitis, periodontal disease),mucositis, musculoskeletal system inflammation (myositis), nonalcoholicsteatohepatitis (nonalcoholic fatty liver disease), ocular & orbitalinflammation (including uveitis, optic neuritis, peripheral rheumatoidulceration, peripheral corneal inflammation,), osteoarthritis,osteomyelitis, pharyngeal inflammation, polyarthritis, proctitis,psoriasis, radiation injury, sarcoidosis, sickle cell necropathy,superficial thrombophlebitis, systemic inflammatory response syndrome,thyroiditis, systemic lupus erythematosus, graft versus host disease,acute burn injury, Behcet's syndrome, Sjögren's syndrome.

The terms patient and subject include human and veterinary subjects.

Human Anti-MAdCAM Antibodies and Characterization Thereof

In one embodiment, the invention provides anti-MAdCAM antibodiescomprising human CDR sequences. In a preferred embodiment, the inventionprovides human anti-MAdCAM antibodies. In some embodiments, humananti-MAdCAM antibodies are produced by immunizing a non-human transgenicanimal, e.g., a rodent, whose genome comprises human immunoglobulingenes so that the transgenic animal produces human antibodies. In someembodiments, the invention provides an anti-MAdCAM antibody that doesnot bind complement.

In a preferred embodiment, the anti-MAdCAM antibody is 1.7.2, 1.8.2,6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4,9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Inanother preferred embodiment, the anti-MAdCAM antibody comprises a lightchain comprising an amino acid sequence selected from SEQ ID NO: 4, 8,12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66 or 68 (with orwithout the signal sequence) or the variable region of any one of saidamino acid sequences, or one or more CDRs from these amino acidsequences. In another preferred embodiment, the anti-MAdCAM antibodycomprises a heavy chain comprising an amino acid sequence selected fromSEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60 or64 (with or without the signal sequence) or the amino acid sequence ofthe variable region, or of one or more CDRs from said amino acidsequences. Also included in the invention are human anti-MAdCAMantibodies comprising the amino acid sequence from the beginning of theCDR1 to the end of the CDR3 of any one of the above-mentioned sequences.The invention further provides an anti-MAdCAM antibody comprising one ormore FR regions of any of the above-mentioned sequences.

The invention further provides an anti-MAdCAM antibody comprising one ofthe afore-mentioned amino acid sequences in which one or moremodifications have been made. In some embodiments, cysteines in theantibody, which may be chemically reactive, are substituted with anotherresidue, such as, without limitation, alanine or serine. In oneembodiment, the substitution is at a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. In some embodiments,the cysteine is canonical.

In some embodiments, an amino acid substitution is made to eliminatepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the heterogeneity in the antibodyproduct. In some embodiments, asparagine-glycine pairs, which formpotential deamidation sites, are eliminated by altering one or both ofthe residues. In some embodiments, an amino acid substitution is made toadd or to remove potential glycosylation sites in the variable region ofan antibody of the invention.

In some embodiments, the C-terminal lysine of the heavy chain of theanti-MAdCAM antibody of the invention is cleaved. In various embodimentsof the invention, the heavy and light chains of the anti-MAdCAMantibodies may optionally include a signal sequence.

In one aspect, the invention provides twelve inhibitory humananti-MAdCAM monoclonal antibodies and the hybridoma cell lines thatproduce them. Table 1 lists the sequence identifiers (SEQ ID NO:) of thenucleic acids encoding the full-length heavy and light chains (includingsignal sequence), and the corresponding full-length deduced amino acidsequences.

TABLE 1 HUMAN ANTI-MAdCAM ANTIBODIES SEQUENCE IDENTIFIER (SEQ ID NO:)Full Length Monoclonal Heavy Light Antibody DNA Protein DNA Protein1.7.2 1 2 3 4 1.8.2 5 6 7 8 6.14.2 9 10 11 12 6.22.2 13 14 15 16 6.34.217 18 19 20 6.67.1 21 22 23 24 6.73.2 25 26 27 28 6.77.1 29 30 31 327.16.6 33 34 35 36 7.20.5 37 38 39 40 7.26.4 41 42 43 44 9.8.2 45 46 4748

In another aspect, the invention provides a modified version of certainof the above-identified human anti-MAdCAM monoclonal antibodies. Table 2lists the sequence identifiers for the DNA and protein sequences of themodified antibodies.

TABLE 2 HUMAN ANTI-MAdCAM ANTIBODIES SEQUENCE IDENTIFIER (SEQ ID NO:)Modified Full Length Monoclonal Heavy Light Antibody DNA Protein DNAProtein 6.22.2-mod 51 52 53 54 6.34.2-mod 55 56 57 58 6.67.1-mod 59 6061 62 6.77.1-mod 63 64 65 66 7.26.4-mod 41 42 67 68

Class and Subclass of Anti-MAdCAM Antibodies

The antibody may be an IgG, an IgM, an IgE, an IgA or an IgD molecule.In a preferred embodiment, the antibody is an IgG class and is an IgG₁,IgG₂, IgG₃ or IgG₄ subclass. In a more preferred embodiment, theanti-MAdCAM antibody is subclass IgG₂ or IgG₄. In another preferredembodiment, the anti-MAdCAM antibody is the same class and subclass asantibody 1.7.2, 1.8.2, 7.16.6, 7.20.5, 7.26.4, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod or 7.26.4-mod which is IgG₂, or 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1 or 9.8.2, which is IgG₄.

The class and subclass of anti-MAdCAM antibodies may be determined byany method known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies are availablecommercially. ELISA, Western Blot as well as other techniques candetermine the class and subclass. Alternatively, the class and subclassmay be determined by sequencing all or a portion of the constant domainsof the heavy and/or light chains of the antibodies, comparing theiramino acid sequences to the known amino acid sequences of variousclasses and subclasses of immunoglobulins, and determining the class andsubclass of the antibodies as the class showing the highest sequenceidentity.

Species and Molecule Selectivity

In another aspect of the invention, the anti-MAdCAM antibodydemonstrates both species and molecule selectivity. In one embodiment,the anti-MAdCAM antibody binds to human, cynomolgus or dog MAdCAM. Insome embodiments, the anti-MAdCAM antibody does not bind to a New Worldmonkey species such as a marmoset. Following the teachings of thespecification, one may determine the species selectivity for theanti-MAdCAM antibody using methods well known in the art. For instance,one may determine species selectivity using Western blot, FACS, ELISA orimmunohistochemistry. In a preferred embodiment, one may determine thespecies selectivity using immunohistochemistry.

In some embodiments, an anti-MAdCAM antibody that specifically bindsMAdCAM has selectivity for MAdCAM over VCAM, fibronectin or any otherantigen that is at least 10 fold, preferably at least 20, 30, 40, 50,60, 70, 80 or 90 fold, most preferably at least 100 fold. In a preferredembodiment, the anti-MAdCAM antibody does not exhibit any appreciablebinding to VCAM, fibronectin or any other antigen other than MAdCAM. Onemay determine the selectivity of the anti-MAdCAM antibody for MAdCAMusing methods well known in the art following the teachings of thespecification. For instance, one may determine the selectivity usingWestern blot, FACS, ELISA, or immunohistochemistry.

Binding Affinity of Anti-MAdCAM Antibodies to MAdCAM

In another aspect of the invention, the anti-MAdCAM antibodiesspecifically bind to MAdCAM with high affinity. In one embodiment, theanti-MAdCAM antibody specifically binds to MAdCAM with a K_(d) of 3×10⁻⁸M or less, as measured by surface plasmon resonance, such as BIAcore. Inmore preferred embodiments, the antibody specifically binds to MAdCAMwith a K_(d) of 1×10⁻⁸ or less or 1×10⁻⁹ M or less. In an even morepreferred embodiment, the antibody specifically binds to MAdCAM with aK_(d) or 1×10⁻¹⁹ M or less. In other preferred embodiments, an antibodyof the invention specifically binds to MAdCAM with a K_(d) of2.66×10⁻′⁹M or less, 2.35×10⁻¹¹M or less or 9×10⁻¹²M or less. In anotherpreferred embodiment, the antibody specifically binds to MAdCAM with aK_(d) or 1×10⁻¹¹ M or less. In another preferred embodiment, theantibody specifically binds to MAdCAM with substantially the same K_(d)as an antibody selected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2,6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod,6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. An antibody with“substantially the same K_(d)” as a reference antibody has a K_(d) thatis ±100 pM, preferably ±50 pM, more preferably ±20 pM, still morepreferably ±10 pM, ±5 pM or ±2 pM, compared to the K_(d) of thereference antibody in the same experiment. In another preferredembodiment, the antibody binds to MAdCAM with substantially the sameK_(d) as an antibody that comprises one or more variable domains or oneor more CDRs from an antibody selected from 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In stillanother preferred embodiment, the antibody binds to MAdCAM withsubstantially the same K_(d) as an antibody that comprises one of theamino acid sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 52,54, 56, 58, 62, 64, 66 or 68 (with or without the signal sequence), orthe variable domain thereof. In another preferred embodiment, theantibody binds to MAdCAM with substantially the same K_(d) as anantibody that comprises one or more CDRs from an antibody that comprisesan amino acid sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 52,54, 56, 58, 62, 64, 66 or 68.

The binding affinity of an anti-MAdCAM antibody to MAdCAM may bedetermined by any method known in the art. In one embodiment, thebinding affinity can be measured by competitive ELISAs, RIAs or surfaceplasmon resonance, such as BIAcore. In a more preferred embodiment, thebinding affinity is measured by surface plasmon resonance. In an evenmore preferred embodiment, the binding affinity and dissociation rate ismeasured using a BIAcore. An example of determining binding affinity isdescribed below in Example II.

Half-Life of Anti-MAdCAM Antibodies

According to another object of the invention, the anti-MAdCAM antibodyhas a half-life of at least one day in vitro or in vivo. In a preferredembodiment, the antibody or portion thereof has a half-life of at leastthree days. In a more preferred embodiment, the antibody or portionthereof has a half-life of four days or longer. In another embodiment,the antibody or portion thereof has a half-life of eight days or longer.In another embodiment, the antibody or antigen-binding portion thereofis derivatized or modified such that it has a longer half-life, asdiscussed below. In another preferred embodiment, the antibody maycontain point mutations to increase serum half life, such as describedWO 00/09560, published Feb. 24, 2000.

The antibody half-life may be measured by any means known to one havingordinary skill in the art. For instance, the antibody half life may bemeasured by Western blot, ELISA or RIA over an appropriate period oftime. The antibody half-life may be measured in any appropriate animal,such as a primate, e.g., cynomolgus monkey, or a human.

Identification of MAdCAM Epitopes Recognized by Anti-MAdCAM Antibody

The invention also provides a human anti-MAdCAM antibody that binds thesame antigen or epitope as a human anti-MAdCAM antibody provided herein.Further, the invention provides a human anti-MAdCAM antibody thatcompetes or cross-competes with a human anti-MAdCAM antibody. In apreferred embodiment, the human anti-MAdCAM antibody is 1.7.2, 1.8.2,6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4,9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Inanother preferred embodiment, the human anti-MAdCAM antibody comprisesone or more variable domains or one or more CDRs from an antibodyselected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In still another preferredembodiment, the human anti-MAdCAM antibody comprises one of the aminoacid sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 52, 54, 56,58, 62, 64, 66 or 68 (with or without the signal sequence), or avariable domain thereof. In another preferred embodiment, the humananti-MAdCAM antibody comprises one or more CDRs from an antibody thatcomprises one of the amino acid sequences selected from SEQ ID NOS: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46 48, 52, 54, 56, 58, 62, 64, 66 or 68. In a highly preferredembodiment, the anti-MAdCAM antibody is another human antibody.

One may determine whether an anti-MAdCAM antibody binds to the sameantigen as another anti-MAdCAM antibody using a variety of methods knownin the art. For instance, one can use a known anti-MAdCAM antibody tocapture the antigen, elute the antigen from the anti-MAdCAM antibody,and then determine whether the test antibody will bind to the elutedantigen. One may determine whether an antibody competes with ananti-MAdCAM antibody by binding the anti-MAdCAM antibody to MAdCAM undersaturating conditions, and then measuring the ability of the testantibody to bind to MAdCAM. If the test antibody is able to bind to theMAdCAM at the same time as the anti-MAdCAM antibody, then the testantibody binds to a different epitope than the anti-MAdCAM antibody.However, if the test antibody is not able to bind to the MAdCAM at thesame time, then the test antibody competes with the human anti-MAdCAMantibody. This experiment may be performed using ELISA, or surfaceplasmon resonance or, preferably, BIAcore. To test whether ananti-MAdCAM antibody cross-competes with another anti-MAdCAM antibody,one may use the competition method described above in two directions,i.e. determining if the known antibody blocks the test antibody and viceversa.

Light and Heavy Chain Gene Usage

The invention also provides an anti-MAdCAM antibody that comprises alight chain variable region encoded by a human κ gene. In a preferredembodiment, the light chain variable region is encoded by a human Vκ A2,A3, A26, B3, O12 or O18 gene family. In various embodiments, the lightchain comprises no more than eleven, no more than six or no more thanthree amino acid substitutions from the germline human Vκ A2, A3, A26,B3, O12 or O18 sequence. In a preferred embodiment, the amino acidsubstitutions are conservative substitutions.

SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 provide theamino acid sequences of the full-length kappa light chains of twelveanti-MAdCAM antibodies, 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1,6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2. FIGS. 1K-1T arealignments of the amino acid sequences of the light chain variabledomains of twelve anti-MAdCAM antibodies with the germline sequencesfrom which they are derived. FIG. 2A shows an alignment of the aminoacid sequences of the light chain variable domains of the kappa lightchains of twelve anti-MAdCAM antibodies to each other. Following theteachings of this specification, one of ordinary skill in the art coulddetermine the differences between the germline sequences and theantibody sequences of additional anti-MAdCAM antibodies. SEQ ID NOS: 54,58, 62, 66 or 68 provide the amino acid sequences of the full lengthkappa light chains of five additional anti-MAdCAM antibodies,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod, modifiedby amino acid substitution from their parent anti-MAdCAM antibodies,6.22.2, 6.34.2, 6.67.1, 6.77.1 or 7.26.4, respectively.

In a preferred embodiment, the VL of the anti-MAdCAM antibody containsthe same mutations, relative to the germline amino acid sequence, as anyone or more of the VL of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Theinvention includes an anti-MAdCAM antibody that utilizes the same humanVκ and human Jk genes as an exemplified antibody. In some embodiments,the antibody comprises one or more of the same mutations from germlineas one or more exemplified antibodies. In some embodiments, the antibodycomprises different substitutions at one or more of the same positionsas one or more of the exemplified antibodies. For example, the VL of theanti-MAdCAM antibody may contain one or more amino acid substitutionsthat are the same as those present in antibody 7.16.6, and another aminoacid substitution that is the same as antibody 7.26.4. In this manner,one can mix and match different features of antibody binding in order toalter, e.g., the affinity of the antibody for MAdCAM or its dissociationrate from the antigen. In another embodiment, the mutations are made inthe same position as those found in any one or more of the VL ofantibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1,7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod,6.77.1-mod or 7.26.4-mod, but conservative amino acid substitutions aremade rather than using the same amino acid. For example, if the aminoacid substitution compared to the germline in one of the antibodies1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6,7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or7.26.4-mod is glutamate, one may conservatively substitute aspartate.Similarly, if the amino acid substitution is serine, one mayconservatively substitute threonine.

In another preferred embodiment, the light chain comprises an amino acidsequence that is the same as the amino acid sequence of the VL of 1.7.2,1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5,7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or7.26.4-mod. In another highly preferred embodiment, the light chaincomprises amino acid sequences that are the same as the CDR regions ofthe light chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment,the light chain comprises an amino acid sequence with at least one CDRregion of the light chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2,6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod,6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferredembodiment, the light chain comprises amino acid sequences with CDRsfrom different light chains that use the same V_(K) and J_(K) genes. Ina more preferred embodiment, the CDRs from different light chains areobtained from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment,the light chain comprises an amino acid sequence selected from SEQ IDNOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 64, 66 or68 with or without the signal sequence. In another embodiment, the lightchain comprises an amino acid sequence encoded by a nucleotide sequenceselected from SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47,53, 57, 61, 65 or 67 (with or without the signal sequence), or anucleotide sequence that encodes an amino acid sequence having 1-11amino acid insertions, deletions or substitutions therefrom. Preferably,the amino acid substitutions are conservative amino acid substitutions.In another embodiment, the antibody or portion thereof comprises alambda light chain.

The present invention also provides an anti-MAdCAM antibody or portionthereof that comprises a human VH gene sequence or a sequence derivedfrom a human VH gene. In one embodiment, the heavy chain amino acidsequence is derived from a human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33or 4-4 gene family. In various embodiments, the heavy chain comprises nomore than fifteen, no more than six or no more than three amino acidchanges from germline human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33 or 4-4gene sequence.

SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 and 46 provide theamino acid sequences of the full-length heavy chains of twelveanti-MAdCAM antibodies. FIGS. 1A-1J are alignments of the amino acidsequences of the heavy chain variable regions of twelve anti-MAdCAMantibodies with the germline sequences from which they are derived. FIG.2B shows the alignments of the amino acid sequences of the heavy chainvariable regions of twelve anti-MAdCAM antibodies to each other.Following the teachings of this specification and the nucleotidesequences of the invention, one of ordinary skill in the art coulddetermine the encoded amino acid sequence of the twelve anti-MAdCAMheavy chains and the germline heavy chains and determine the differencesbetween the germline sequences and the antibody sequences. SEQ ID NOS:52, 56, 60 and 64 provide the amino acid sequences of the full lengthheavy chains of anti-MAdCAM antibodies, 6.22.2-mod, 6.34.2-mod and6.67.1-mod, modified by amino acid substitution from their parentanti-MAdCAM antibodies, 6.22.2, 6.34.2 and 6.67.1 respectively. Onefurther modified anti-MAdCAM antibody, 7.26.4-mod, has a full lengthheavy chain amino acid sequence which is SEQ ID NO: 42.

In a preferred embodiment, the VH of the anti-MAdCAM antibody containsthe same mutations, relative to the germline amino acid sequence, as anyone or more of the VH of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Similar tothat discussed above, the antibody comprises one or more of the samemutations from germline as one or more exemplified antibodies. In someembodiments, the antibody comprises different substitutions at one ormore of the same positions as one or more of the exemplified antibodies.For example, the VH of the anti-MAdCAM antibody may contain one or moreamino acid substitutions that are the same as those present in antibody7.16.6, and another amino acid substitution that is the same as antibody7.26.4. In this manner, one can mix and match different features ofantibody binding in order to alter, e.g., the affinity of the antibodyfor MAdCAM or its dissociation rate from the antigen. In anotherembodiment, an amino acid substitution compared to germline is made atthe same position as a substitution from germline as found in any one ormore of the VH of reference antibody 1.7.2, 1.8.2, 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod, but theposition is substituted with a different residue, which is aconservative substitution compared to the reference antibody.

In another preferred embodiment, the heavy chain comprises an amino acidsequence that is the same as the amino acid sequence of the VH of 1.7.2,1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5,7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or7.26.4-mod. In another highly preferred embodiment, the heavy chaincomprises amino acid sequences that are the same as the CDR regions ofthe heavy chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment,the heavy chain comprises an amino acid sequence from at least one CDRregion of the heavy chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2,6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.4, 7.26.4, 9.8.2, 6.22.2-mod,6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferredembodiment, the heavy chain comprises amino acid sequences with CDRsfrom different heavy chains. In a more preferred embodiment, the CDRsfrom different heavy chains are obtained from 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In anotherpreferred embodiment, the heavy chain comprises an amino acid sequenceselected from SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46,52, 56, 60 or 64 with or without the signal sequence. In anotherembodiment, the heavy chain comprises an amino acid sequence encoded bya nucleotide sequence selected from SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25,29, 33, 37, 41, 45, 51, 55, 59 or 63, or a nucleotide sequence thatencodes an amino acid sequence having 1-15 amino acid insertions,deletions or substitutions therefrom. In another embodiment, thesubstitutions are conservative amino acid substitutions.

Methods of Producing Antibodies and Antibody-Producing Cell LinesImmunization

In one embodiment of the instant invention, human antibodies areproduced by immunizing a non-human animal comprising some or all of thehuman immunoglobulin heavy and light chain loci with an MAdCAM antigen.In a preferred embodiment, the non-human animal is a XENOMOUSE™ animal,which is an engineered mouse strain that comprises large fragments ofthe human immunoglobulin loci and is deficient in mouse antibodyproduction. See, e.g., Green et al., Nature Genetics 7:13-21 (1994) andU.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181,6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741, WO 94/02602,WO 96/34096 and WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO99/45031, WO 99/53049, WO 00 09560 and WO 00/037504. The XENOMOUSE™animal produces an adult-like human repertoire of fully human antibodiesand generates antigen-specific human mAbs. A second generationXENOMOUSE™ animal contains approximately 80% of the human antibody Vgene repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and κ lightchain loci. In other embodiments, XENOMOUSE™ mice contain approximatelyall of the human heavy chain and λ light chain locus. See Mendez et al.,Nature Genetics 15:146-156 (1997), Green and Jakobovits, J. Exp. Med.188:483-495 (1998), the disclosures of which are hereby incorporated byreference.

The invention also provides a method for making anti-MAdCAM antibodiesfrom non-human, non-mouse animals by immunizing non-human transgenicanimals that comprise human immunoglobulin loci. One may produce suchanimals using the methods described immediately above. The methodsdisclosed in these documents can be modified as described in U.S. Pat.No. 5,994,619 (the “'619 patent”), which is here in incorporated byreference. The '619 patent describes methods for producing novelcultured inner cell mass (CICM) cells and cell lines, derived from pigsand cows, and transgenic CICM cells into which heterologous DNA has beeninserted. CICM transgenic cells can be used to produce cloned transgenicembryos, fetuses, and offspring. The '619 patent also describes methodsof producing transgenic animals that are capable of transmitting theheterologous DNA to their progeny. In a preferred embodiment, thenon-human animals may be rats, sheep, pigs, goats, cattle or horses.

In another embodiment, the non-human animal comprising humanimmunoglobulin loci are animals that have a “minilocus” of humanimmunoglobulins. In the minilocus approach, an exogenous Ig locus ismimicked through the inclusion of individual genes from the Ig locus.Thus, one or more VH genes, one or more DH genes, one or more JH genes,a μ, constant domain(s), and a second constant domain(s) (preferably agamma constant domain(s) are formed into a construct for insertion intoan animal. This approach is described, inter alia, in U.S. Pat. Nos.5,545,807, 5,545,806, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and5,643,763, hereby incorporated by reference.

An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. However, a potential disadvantage of theminilocus approach is that there may not be sufficient immunoglobulindiversity to support full B-cell development, such that there may belower antibody production.

To produce a human anti-MAdCAM antibody, a non-human animal comprisingsome or all of the human immunoglobulin loci is immunized with a MAdCAMantigen and an antibody or the antibody-producing cell is isolated fromthe animal. The MAdCAM antigen may be isolated and/or purified MAdCAMand is preferably a human MAdCAM. In another embodiment, the MAdCAMantigen is a fragment of MAdCAM, preferably the extracellular domain ofMAdCAM. In another embodiment, the MAdCAM antigen is a fragment thatcomprises at least one epitope of MAdCAM. In another embodiment, theMAdCAM antigen is a cell that expresses MAdCAM on its cell surface,preferably a cell that overexpresses MAdCAM on its cell surface.

Immunization of animals may be done by any method known in the art.

See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York:Cold Spring Harbor Press (1990). Methods for immunizing non-humananimals such as mice, rats, sheep, goats, pigs, cattle and horses arewell known in the art. See, e.g., Harlow and Lane and U.S. Pat. No.5,994,619. In a preferred embodiment, the MAdCAM antigen is administeredwith an adjuvant to stimulate the immune response. Such adjuvantsinclude complete or incomplete Freund's adjuvant, RIBI (muramyldipeptides) or ISCOM (immunostimulating complexes). Such adjuvants mayprotect the polypeptide from rapid dispersal by sequestering it in alocal deposit, or they may contain substances that stimulate the host tosecrete factors that are chemotactic for macrophages and othercomponents of the immune system. Preferably, if a polypeptide is beingadministered, the immunization schedule will involve two or moreadministrations of the polypeptide, spread out over several weeks.

Example I provides a protocol for immunizing a XENOMOUSE™ animal withfull-length human MAdCAM in phosphate-buffered saline.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a MAdCAM antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-MAdCAMantibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-MAdCAM antibodies may be purified from the serum.

In another embodiment, antibody-producing immortalized cell lines may beprepared from the immunized animal. After immunization, the animal issacrificed and B cells are immortalized using methods well-known in theart. Methods of immortalizing cells include, but are not limited to,transfecting them with oncogenes, infecting them with an oncogenic virusand cultivating them under conditions that select for immortalizedcells, subjecting them to carcinogenic or mutating compounds, fusingthem with an immortalized cell, e.g., a myeloma cell, and inactivating atumor suppressor gene. See, e.g., Harlow and Lane, supra. In embodimentsinvolving the myeloma cells, the myeloma cells do not secreteimmunoglobulin polypeptides (a non-secretory cell line). Afterimmortalization and antibiotic selection, the immortalized cells, orculture supernatants thereof, are screened using MAdCAM, a portionthereof, or a cell expressing MAdCAM. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example ofELISA screening is provided in PCT Publication No. WO 00/37504, hereinincorporated by reference.

In another embodiment, antibody-producing cells may be prepared from ahuman who has an autoimmune disorder and who expresses anti-MAdCAMantibodies. Cells expressing the anti-MAdCAM antibodies may be isolatedby isolating white blood cells and subjecting them tofluorescence-activated cell sorting (FACS) or by panning on platescoated with MAdCAM or a portion thereof. These cells may be fused with ahuman non-secretory myeloma to produce human hybridomas expressing humananti-MAdCAM antibodies. In general, this is a less preferred embodimentbecause it is likely that the anti-MAdCAM antibodies will have a lowaffinity for MAdCAM.

Anti-MAdCAM antibody-producing cells, e.g., hybridomas are selected,cloned and further screened for desirable characteristics, includingrobust cell growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

Preferably, the immunized animal is a non-human animal that expresseshuman immunoglobulin genes and the splenic B cells are fused to amyeloma derived from the same species as the non-human animal Morepreferably, the immunized animal is a XENOMOUSE™ animal and the myelomacell line is a non-secretory mouse myeloma, such as the myeloma cellline is P3-X63-AG8-653 (ATCC). See, e.g., Example I.

Thus, in one embodiment, the invention provides methods for producing acell line that produces a human monoclonal antibody or a fragmentthereof directed to MAdCAM comprising (a) immunizing a non-humantransgenic animal described herein with MAdCAM, a portion of MAdCAM or acell or tissue expressing MAdCAM; (b) allowing the transgenic animal tomount an immune response to MAdCAM; (c) isolating antibody-producingcells from transgenic animal; (d) immortalizing the antibody-producingcells; (e) creating individual monoclonal populations of theimmortalized antibody-producing cells; and (f) screening theimmortalized antibody-producing cells or culture supernatants thereof toidentify an antibody directed to MAdCAM.

In one aspect, the invention provides hybridomas that produce humananti-MAdCAM antibodies. In a preferred embodiment, the hybridomas aremouse hybridomas, as described above. In another embodiment, thehybridomas are produced in a non-human, non-mouse species such as rats,sheep, pigs, goats, cattle or horses. In another embodiment, thehybridomas are human hybridomas, in which a human non-secretory myelomais fused with a human cell expressing an anti-MAdCAM antibody.

Nucleic Acids, Vectors, Host Cells and Recombinant Methods of MakingAntibodies Nucleic Acids

Nucleic acid molecules encoding anti-MAdCAM antibodies of the inventionare provided. In one embodiment, the nucleic acid molecule encodes aheavy and/or light chain of an anti-MAdCAM immunoglobulin. In apreferred embodiment, a single nucleic acid molecule encodes a heavychain of an anti-MAdCAM immunoglobulin and another nucleic acid moleculeencodes the light chain of an anti-MAdCAM immunoglobulin. In a morepreferred embodiment, the encoded immunoglobulin is a humanimmunoglobulin, preferably a human IgG. The encoded light chain may be aλ chain or a κ chain, preferably a κ chain.

In a preferred embodiment the nucleic acid molecule encoding thevariable region of the light chain comprises the germline sequence of ahuman Vκ the A2, A3, A26, B3, O12 or O18 gene or a variant of saidsequence. In a preferred embodiment, the nucleic acid molecule encodingthe light chain comprises a sequence derived from a human Jκ1, Jκ2, Jκ3,Jκ4 or Jκ5 gene. In a preferred embodiment, the nucleic acid moleculeencoding the light chain encodes no more than eleven amino acid changesfrom the germline A2, A3, A26, B3, O12 or O18 Vκ gene, preferably nomore than six amino acid changes, and even more preferably no more thanthree amino acid changes. In a more preferred embodiment, the nucleicacid encoding the light chain is the germline sequence.

The invention provides a nucleic acid molecule that encodes a variableregion of the light chain (VL) containing up to eleven amino acidchanges compared to the germline sequence, wherein the amino acidchanges are identical to amino acid changes from the germline sequencefrom the VL of one of the antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Theinvention also provides a nucleic acid molecule comprising a nucleotidesequence that encodes the amino acid sequence of the variable region ofthe light chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The invention also provides anucleic acid molecule comprising a nucleotide sequence that encodes theamino acid sequence of one or more of the CDRs of any one of the lightchains of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1,7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod,6.77.1-mod or 7.26.4-mod. In a preferred embodiment, the nucleic acidmolecule comprises a nucleotide sequence that encodes the amino acidsequence of all of the CDRs of any one of the light chains of 1.7.2,1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5,7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or7.26.4-mod. In another embodiment, the nucleic acid molecule comprises anucleotide sequence that encodes the amino acid sequence of one of SEQID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68or comprises a nucleotide sequence of one of SEQ ID NOS: 3, 7, 11, 15,19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67. In anotherpreferred embodiment, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one or more of the CDRsof any one of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48,54, 58, 62, 66, 68 or comprises a nucleotide sequence of one or more ofthe CDRs of any one of SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39,43, 47, 53, 57, 61, 65, or 67. In a more preferred embodiment, thenucleic acid molecule comprises a nucleotide sequence that encodes theamino acid sequence of all of the CDRs of any one of SEQ ID NOS: 4, 8,12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68 or comprisesa the nucleotide sequence of all the CDRs of any one of SEQ ID NOS: 3,7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65, or 67.

The invention also provides a nucleic acid molecule that encodes anamino acid sequence of a VL that has an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to aVL described above, particularly to a VL that comprises an amino acidsequence of one of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44,48, 54, 58, 62, 66 or 68. The invention also provides a nucleotidesequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to a nucleotide sequence of one of SEQ ID NOS: 3, 7, 11,15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67.

In another embodiment, the invention provides a nucleic acid moleculethat hybridizes under highly stringent conditions to a nucleic acidmolecule encoding a VL as described above, particularly a nucleic acidmolecule that comprises a nucleotide sequence encoding an amino acidsequence of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48,54, 58, 62, 66 or 68. The invention also provides a nucleic acidmolecule that hybridizes under highly stringent conditions to a nucleicacid molecule comprising a nucleotide sequence of one of SEQ ID NOS: 3,7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67.

The invention also provides a nucleic acid molecule encoding a heavychain variable region (VH) that utilizes a human VH 1-18, 3-15, 3-21,3-23, 3-30, 3-33 or 4-4 VH gene. In some embodiments, the nucleic acidmolecule encoding the VH gene further utilizes a human JH4 or JH6 familygene. In some embodiments, the nucleic acid molecule encoding the VHgene utilize the human JH4b or JH6b gene. In another embodiment, thenucleic acid molecule comprises a sequence derived from a human D 3-10,4-23, 5-5, 6-6 or 6-19 gene. In an even more preferred embodiment, thenucleic acid molecule encoding the VH contains no more than fifteenamino acid changes from the germline VH 1-18, 3-15, 3-21, 3-23, 3-30,3-33 or 4-4 genes, preferably no more than six amino acid changes, andeven more preferably no more than three amino acid changes. In a highlypreferred embodiment, the nucleic acid molecule encoding the VH containsat least one amino acid change compared to the germline sequence,wherein the amino acid change is identical to an amino acid change fromthe germline sequence from the heavy chain of one of the antibodies1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6,7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or7.26.4-mod. In an even more preferred embodiment, the VH contains nomore than fifteen amino acid changes compared to the germline sequences,wherein the changes are identical to those changes from the germlinesequence from the VH of one of the antibodies 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod.

In one embodiment, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of the VH of 1.7.2, 1.8.2,6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4,9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Inanother embodiment, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one or more of the CDRsof the heavy chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1,6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In a preferred embodiment, thenucleic acid molecule comprises nucleotide sequences that encode theamino acid sequences of all of the CDRs of the heavy chain of 1.7.2,1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5,7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or7.26.4-mod. In another preferred embodiment, the nucleic acid moleculecomprises a nucleotide sequence that encodes the amino acid sequence ofone of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56,60 or 64 or that comprises a nucleotide sequence of one of SEQ ID NOS:1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59 or 63. Inanother preferred embodiment, the nucleic acid molecule comprises anucleotide sequence that encodes the amino acid sequence of one or moreof the CDRs of any one of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34,38, 42, 46, 52, 56, 60 or 64 or comprises a nucleotide sequence of oneor more of the CDRs of any one of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25,29, 33, 37, 41, 45, 51, 55, 59 or 63. In a preferred embodiment, thenucleic acid molecule comprises a nucleotide sequence that encodes theamino acid sequences of all of the CDRs of any one of SEQ ID NOS: 2, 6,10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60 or 64 or comprises anucleotide sequence of all of the CDRs of any one of SEQ ID NOS: 1, 5,9, 13, 17, 21, 25, 29, 33, 37, 41 45, 51, 55, 59 or 63. In someembodiments the nucleic acid molecule comprises a nucleotide sequenceencoding a contiguous region from the beginning of CDR1 to the end ofCDR3 of a heavy or light chain of any of the above-mentioned anti-MAdCAMantibodies.

In another embodiment, the nucleic acid molecule encodes an amino acidsequence of a VH that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to one of the amino acid sequences encoding aVH as described immediately above, particularly to a VH that comprisesan amino acid sequence of one of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26,30, 34, 38, 42, 46, 52, 56, 60 or 64. The invention also provides anucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to a nucleotide sequence of one of SEQ ID NOS:1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59 or 63.

In another embodiment, the nucleic acid molecule encoding a VH is onethat hybridizes under highly stringent conditions to a nucleotidesequence encoding a VH as described above, particularly to a VH thatcomprises an amino acid sequence of one of SEQ ID NOS: 2, 6, 10, 14, 18,22, 26, 30, 34, 38, 42, 46, 52, 56, 60 or 64. The invention alsoprovides a nucleotide sequence encoding a VH that hybridizes underhighly stringent conditions to a nucleic acid molecule comprising anucleotide sequence of one of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29,33, 37, 41, 45, 51, 55, 59 or 63.

The nucleotide sequence encoding either or both of the entire heavy andlight chains of an anti-MAdCAM antibody or the variable regions thereofmay be obtained from any source that produces an anti-MAdCAM antibody.Methods of isolating mRNA encoding an antibody are well-known in theart. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989). The mRNA may be used to produce cDNA for use in the polymerasechain reaction (PCR) or cDNA cloning of antibody genes. In oneembodiment of the invention, the nucleic acid molecules may be obtainedfrom a hybridoma that expresses an anti-MAdCAM antibody, as describedabove, preferably a hybridoma that has as one of its fusion partners atransgenic animal cell that expresses human immunoglobulin genes, suchas a XENOMOUSE™ animal, a non-human mouse transgenic animal or anon-human, non-mouse transgenic animal. In another embodiment, thehybridoma is derived from a non-human, non-transgenic animal, which maybe used, e.g., for humanized antibodies.

A nucleic acid molecule encoding the entire heavy chain of ananti-MAdCAM antibody may be constructed by fusing a nucleic acidmolecule encoding the entire variable domain of a heavy chain or anantigen-binding domain thereof with a constant domain of a heavy chain.Similarly, a nucleic acid molecule encoding the light chain of ananti-MAdCAM antibody may be constructed by fusing a nucleic acidmolecule encoding the variable domain of a light chain or anantigen-binding domain thereof with a constant domain of a light chain.Nucleic acid molecules encoding the VH and VL regions may be convertedto full-length antibody genes by inserting them into expression vectorsalready encoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to theheavy chain constant region (CH) segment(s) within the vector and the VLsegment is operatively linked to the light chain constant region (CL)segment within the vector. Alternatively, the nucleic acid moleculesencoding the VH or VL chains are converted into full-length antibodygenes by linking, e.g., ligating, the nucleic acid molecule encoding aVH chain to a nucleic acid molecule encoding a CH chain using standardmolecular biological techniques. The same may be achieved using nucleicacid molecules encoding VL and CL chains. The sequences of human heavyand light chain constant region genes are known in the art. See, e.g.,Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.,NIH Publ. No. 91-3242 (1991). Nucleic acid molecules encoding thefull-length heavy and/or light chains may then be expressed from a cellinto which they have been introduced and the anti-MAdCAM antibodyisolated.

In a preferred embodiment, the nucleic acid encoding the variable regionof the heavy chain encodes the variable region of amino acid sequencesof SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60or 64, and the nucleic acid molecule encoding the variable region of thelight chains encodes the variable region of amino acid sequence of SEQID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66 or68.

In one embodiment, a nucleic acid molecule encoding either the heavychain of an anti-MAdCAM antibody or an antigen-binding portion thereof,or the light chain of an anti-MAdCAM antibody or an antigen-bindingportion thereof may be isolated from a non-human, non-mouse animal thatexpresses human immunoglobulin genes and has been immunized with aMAdCAM antigen. In other embodiment, the nucleic acid molecule may beisolated from an anti-MAdCAM antibody-producing cell derived from anon-transgenic animal or from a human patient who produces anti-MAdCAMantibodies. mRNA from the anti-MAdCAM antibody-producing cells may beisolated by standard techniques, cloned and/or amplified using PCR andlibrary construction techniques, and screened using standard protocolsto obtain nucleic acid molecules encoding anti-MAdCAM heavy and lightchains.

The nucleic acid molecules may be used to recombinantly express largequantities of anti-MAdCAM antibodies, as described below. The nucleicacid molecules may also be used to produce chimeric antibodies, singlechain antibodies, immunoadhesins, diabodies, mutated antibodies andantibody derivatives, as described further below. If the nucleic acidmolecules are derived from a non-human, non-transgenic animal, thenucleic acid molecules may be used for antibody humanization, also asdescribed below.

In another embodiment, the nucleic acid molecules of the invention maybe used as probes or PCR primers for specific antibody sequences. Forinstance, a nucleic acid molecule probe may be used in diagnosticmethods or a nucleic acid molecule PCR primer may be used to amplifyregions of DNA that could be used, inter alia, to isolate nucleotidesequences for use in producing variable domains of anti-MAdCAMantibodies. In a preferred embodiment, the nucleic acid molecules areoligonucleotides. In a more preferred embodiment, the oligonucleotidesare from highly variable regions of the heavy and light chains of theantibody of interest. In an even more preferred embodiment, theoligonucleotides encode all or a part of one or more of the CDRs.

Vectors

The invention provides vectors comprising the nucleic acid molecules ofthe invention that encode the heavy chain or the antigen-binding portionthereof. The invention also provides vectors comprising the nucleic acidmolecules of the invention that encode the light chain orantigen-binding portion thereof. The invention also provides vectorscomprising nucleic acid molecules encoding fusion proteins, modifiedantibodies, antibody fragments, and probes thereof.

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. Expression vectors include plasmids, retroviruses,adenoviruses, adeno-associated viruses (AAV), plant viruses such ascauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBVderived episomes, and the like. The antibody gene is ligated into avector such that transcriptional and translational control sequenceswithin the vector serve their intended function of regulating thetranscription and translation of the antibody gene. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. The antibody light chain gene and theantibody heavy chain gene can be inserted into separate vector. In apreferred embodiment, both genes are inserted into the same expressionvector. The antibody genes are inserted into the expression vector bystandard methods (e.g., ligation of complementary restriction sites onthe antibody gene fragment and vector, or blunt end ligation if norestriction sites are present).

A convenient vector is one that encodes a functionally complete human CHor CL immunoglobulin sequence, with appropriate restriction sitesengineered so that any VH or VL sequence can be easily inserted andexpressed, as described above. In such vectors, splicing usually occursbetween the splice donor site in the inserted J region and the spliceacceptor site preceding the human C region, and also at the spliceregions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The recombinant expression vector can also encodea signal peptide that facilitates secretion of the antibody chain from ahost cell. The antibody chain gene may be cloned into the vector suchthat the signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see e.g., U.S. Pat.Nos. 5,168,062, 4,510,245, and 4,968,615, each of which is herebyincorporated by reference. Methods for expressing antibodies in plants,including a description of promoters and vectors, as well astransformation of plants are known in the art. See, e.g, U.S. Pat. No.6,517,529. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection), and the glutamate synthetase gene

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding the heavy chain or an antigen-bindingportion thereof and/or the light chain or an antigen-binding portionthereof of an anti-MAdCAM antibody, and vectors comprising these nucleicacid molecules, can be used for transformation of a suitable mammalianplant, bacterial or yeast host cell. Transformation can be by any knownmethod for introducing polynucleotides into a host cell. Methods forintroduction of heterologous polynucleotides into mammalian cells arewell known in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene-mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, biolistic injection and direct microinjection of the DNA intonuclei. In addition, nucleic acid molecules may be introduced intomammalian cells by viral vectors. Methods of transforming cells are wellknown in the art. See, e.g., U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (which patents are hereby incorporated hereinby reference). Methods of transforming plant cells are well known in theart, including, e.g., Agrobacterium-mediated transformation, biolistictransformation, direct injection, electroporation and viraltransformation. Methods of transforming bacterial and yeast cells arealso well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NS0, SP2 cells, HEK-293T cells,NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, monkeykidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),A549 cells, 3T3 cells, and a number of other cell lines. Mammalian hostcells include human, mouse, rat, dog, monkey, pig, goat, bovine, horseand hamster cells. Cell lines of particular preference are selectedthrough determining which cell lines have high expression levels. Othercell lines that may be used are insect cell lines, such as Sf9 cells,amphibian cells, bacterial cells, plant cells and fungal cells. Whenrecombinant expression vectors encoding the heavy chain orantigen-binding portion thereof, the light chain and/or antigen-bindingportion thereof are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods. Plant host cellsinclude, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato,etc. Bacterial host cells include E. coli and Streptomyces species.Yeast host cells include Schizosaccharomyces pombe, Saccharomycescerevisiae and Pichia pastoris.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase gene expressionsystem (the GS system) is a common approach for enhancing expressionunder certain conditions. The GS system is discussed in whole or part inconnection with European Patent Nos. 0 216 846, 0 256 055, 0 338 841 and0 323 997.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation of theantibodies.

Transgenic Animals and Plants

The invention also provides transgenic non-human animals and transgenicplants comprising one or more nucleic acid molecules of the inventionthat may be used to produce antibodies of the invention. Antibodies canbe produced in and recovered from tissue or bodily fluids, such as milk,blood or urine, of goats, cows, horses, pigs, rats, mice, rabbits,hamsters or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690,5,756,687, 5,750,172, and 5,741,957. As described above, non-humantransgenic animals that comprise human immunoglobulin loci can beimmunized with MAdCAM or a portion thereof. Methods for makingantibodies in plants are described, e.g., in U.S. Pat. Nos. 6,046,037and 5,959,177, incorporated herein by reference.

In another embodiment, non-human transgenic animals and transgenicplants are produced by introducing one or more nucleic acid molecules ofthe invention into the animal or plant by standard transgenictechniques. See Hogan, supra. The transgenic cells used for making thetransgenic animal can be embryonic stem cells, somatic cells orfertilized egg cells. The transgenic non-human organisms can bechimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See,e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual2ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Geneticsand Transgenics: A Practical Approach, Oxford University Press (2000);and Pinkert, Transgenic Animal Technology: A Laboratory Handbook,Academic Press (1999). In another embodiment, the transgenic non-humanorganisms may have a targeted disruption and replacement that encodes aheavy chain and/or a light chain of interest. In a preferred embodiment,the transgenic animals or plants comprise and express nucleic acidmolecules encoding heavy and light chains that combine to bindspecifically to MAdCAM, preferably human MAdCAM. In another embodiment,the transgenic animals or plants comprise nucleic acid moleculesencoding a modified antibody such as a single-chain antibody, a chimericantibody or a humanized antibody. The anti-MAdCAM antibodies may be madein any transgenic animal. In a preferred embodiment, the non-humananimals are mice, rats, sheep, pigs, goats, cattle or horses. Thenon-human transgenic animal expresses said encoded polypeptides inblood, milk, urine, saliva, tears, mucus and other bodily fluids.

Phage Display Libraries

The invention provides a method for producing an anti-MAdCAM antibody orantigen-binding portion thereof comprising the steps of synthesizing alibrary of human antibodies on phage, screening the library with aMAdCAM or a portion thereof, isolating phage that bind MAdCAM, andobtaining the antibody from the phage. One method to prepare the libraryof antibodies comprises the steps of immunizing a non-human host animalcomprising a human immunoglobulin locus with MAdCAM or an antigenicportion thereof to create an immune response, extracting cells from thehost animal the cells that are responsible for production of antibodies;isolating RNA from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using a primer, and inserting the cDNAinto phage display vector such that antibodies are expressed on thephage. Recombinant anti-MAdCAM antibodies of the invention may beobtained in this way.

Recombinant anti-MAdCAM human antibodies of the invention in addition tothe anti-MAdCAM antibodies disclosed herein can be isolated by screeningof a recombinant combinatorial antibody library, preferably a scFv phagedisplay library, prepared using human VL and VH cDNAs prepared from mRNAisolated from human lymphocytes. Methodologies for preparing andscreening such libraries are known in the art. There are commerciallyavailable kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Thereare also other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO92/01047; PCT Publication No. WO 92/09690; Fuchs et al. (1991),Biotechnology, 9:1369-1372; Hay et al., Hum. Antibod. Hybridomas,3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCaffertyet al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J, 12:725-734(1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson etal., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci.USA, 89:3576-3580 (1992); Garrad et al., Biotechnology, 9:1373-1377(1991); Hoogenboom et al., Nuc Acid Res, 19:4133-4137 (1991); and Barbaset al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991).

In a preferred embodiment, to isolate human anti-MAdCAM antibodies withthe desired characteristics, a human anti-MAdCAM antibody as describedherein is first used to select human heavy and light chain sequenceshaving similar binding activity toward MAdCAM, using the epitopeimprinting methods described in Hoogenboom et al., PCT Publication No.WO 93/06213. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described in McCafferty et al.,PCT Publication No. WO 92/01047, McCafferty et al., Nature, 348:552-554(1990); and Griffiths et al., EMBO J, 12:725-734 (1993). The scFvantibody libraries preferably are screened using human MAdCAM as theantigen.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for MAdCAM binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve thequality of the antibody, the VL and VH segments of the preferred VL/VHpair(s) can be randomly mutated, preferably within the CDR3 region of VHand/or VL, in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. This in vitro affinity maturation can beaccomplished by amplifying VH and VL regions using PCR primerscomplimentary to the VH CDR3 or VL CDR3, respectively, which primershave been “spiked” with a random mixture of the four nucleotide bases atcertain positions such that the resultant PCR products encode VH and VLsegments into which random mutations have been introduced into the VHand/or VL CDR3 regions. These randomly mutated VH and VL segments can berescreened for binding to MAdCAM.

Following screening and isolation of an anti-MAdCAM antibody of theinvention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention, as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Class Switching

Another aspect of the instant invention is to provide a mechanism bywhich the class of an anti-MAdCAM antibody may be switched with another.In one aspect of the invention, a nucleic acid molecule encoding VL orVH is isolated using methods well-known in the art such that it does notinclude any nucleotide sequences encoding CL or CH. The nucleic acidmolecule encoding VL or VH is then operatively linked to a nucleotidesequence encoding a CL or CH from a different class of immunoglobulinmolecule. This may be achieved using a vector or nucleic acid moleculethat comprises a CL or CH encoding sequence, as described above. Forexample, an anti-MAdCAM antibody that was originally IgM may be classswitched to an IgG. Further, the class switching may be used to convertone IgG subclass to another, e.g., from IgG₄ to IgG₂. A preferred methodfor producing an antibody of the invention comprising a desired isotypeor antibody subclass comprises the steps of isolating a nucleic acidencoding the heavy chain of an anti-MAdCAM antibody and a nucleic acidencoding the light chain of an anti-MAdCAM antibody, obtaining thevariable region of the heavy chain, ligating the variable region of theheavy chain with the constant domain of a heavy chain of the desiredisotype, expressing the light chain and the ligated heavy chain in acell, and collecting the anti-MAdCAM antibody with the desired isotype.

Antibody Derivatives

One may use the nucleic acid molecules described above to generateantibody derivatives using techniques and methods known to one ofordinary skill in the art.

Humanized Antibodies

The immunogenicity of non-human antibodies can be reduced to some extentusing techniques of humanization, potentially employing displaytechniques using appropriate libraries. It will be appreciated thatmurine antibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See, e.g., Winter andHarris, Immunol Today, 14:43-46 (1993) and Wright et al., Crit. Reviewsin Immunol., 12125-168 (1992). The antibody of interest may beengineered by recombinant DNA techniques to substitute the C_(H)1,C_(H)2, C_(H)3, hinge domains, and/or the framework domain with thecorresponding human sequence (see WO 92/02190 and U.S. Pat. Nos.5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085).In another embodiment, a non-human anti-MAdCAM antibody can be humanizedby substituting the C_(H)1, hinge domain, C_(H)2, C_(H)3, and/or theframework domains with the corresponding human sequence of a anti-MAdCAMantibody of the invention.

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and hostcells may be used to make mutated anti-MAdCAM antibodies. The antibodiesmay be mutated in the variable domains of the heavy and/or light chainsto alter a binding property of the antibody. For example, a mutation maybe made in one or more of the CDR regions to increase or decrease theK_(d) of the antibody for MAdCAM. Techniques in site-directedmutagenesis are well-known in the art. See, e.g., Sambrook et al., andAusubel et al., supra. In a preferred embodiment, mutations are made atan amino acid residue that is known to be changed compared to germlinein a variable region of an anti-MAdCAM antibody. In a more preferredembodiment, one or more mutations are made at an amino acid residue thatis known to be changed compared to the germline in a variable region orCDR region of one of the anti-MAdCAM antibodies 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In anotherembodiment, one or more mutations are made at an amino acid residue thatis known to be changed compared to the germline in a variable region orCDR region whose amino acid sequence is presented in SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 52, 54, 56, 58, 62, 64, 66 or 68, or whose nucleotidesequence is presented in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 53, 55, 57,61, 63, 65 or 67. In another embodiment, the nucleic acid molecules aremutated in one or more of the framework regions. A mutation may be madein a framework region or constant domain to increase the half-life ofthe anti-MAdCAM antibody. See, e.g., WO 00/09560, published Feb. 24,2000, herein incorporated by reference. In one embodiment, there may beone, three or five or ten point mutations and no more than fifteen pointmutations. A mutation in a framework region or constant domain may alsobe made to alter the immunogenicity of the antibody, to provide a sitefor covalent or non-covalent binding to another molecule, or to altersuch properties as complement fixation. Mutations may be made in each ofthe framework regions, the constant domain and the variable regions in asingle mutated antibody. Alternatively, mutations may be made in onlyone of the framework regions, the variable regions or the constantdomain in a single mutated antibody.

In one embodiment, there are no greater than fifteen amino acid changesin either the VH or VL regions of the mutated anti-MAdCAM antibodycompared to the anti-MAdCAM antibody prior to mutation. In a morepreferred embodiment, there is no more than ten amino acid changes ineither the VH or VL regions of the mutated anti-MAdCAM antibody, morepreferably no more than five amino acid changes, or even more preferablyno more than three amino acid changes. In another embodiment, there areno more than fifteen amino acid changes in the constant domains, morepreferably, no more than ten amino acid changes, even more preferably,no more than five amino acid changes.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be madewhich comprises all or a portion of an anti-MAdCAM antibody linked toanother polypeptide. In a preferred embodiment, only the variableregions of the anti-MAdCAM antibody are linked to the polypeptide. Inanother preferred embodiment, the VH domain of an anti-MAdCAM antibodyare linked to a first polypeptide, while the VL domain of an anti-MAdCAMantibody are linked to a second polypeptide that associates with thefirst polypeptide in a manner in which the VH and VL domains caninteract with one another to form an antibody binding site. In anotherpreferred embodiment, the VH domain is separated from the VL domain by alinker such that the VH and VL domains can interact with one another(see below under Single Chain Antibodies). The VH-linker-VL antibody isthen linked to the polypeptide of interest. The fusion antibody isuseful to directing a polypeptide to a MAdCAM-expressing cell or tissue.The polypeptide may be a therapeutic agent, such as a toxin, growthfactor or other regulatory protein, or may be a diagnostic agent, suchas an enzyme that may be easily visualized, such as horseradishperoxidase. In addition, fusion antibodies can be created in which two(or more) single-chain antibodies are linked to one another. This isuseful if one wants to create a divalent or polyvalent antibody on asingle polypeptide chain, or if one wants to create a bispecificantibody.

To create a single chain antibody, (scFv) the VH- and VL-encoding DNAfragments are operatively linked to another fragment encoding a flexiblelinker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃, such thatthe VH and VL sequences can be expressed as a contiguous single-chainprotein, with the VL and VH regions joined by the flexible linker (see,e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc.Natl. Acad. Sci. USA, 85:5879-5883 (1988); McCafferty et al., Nature,348:552-554 (1990)). The single chain antibody may be monovalent, ifonly a single VH and VL are used, bivalent, if two VH and VL are used,or polyvalent, if more than two VH and VL are used.

In another embodiment, other modified antibodies may be prepared usinganti-MAdCAM-encoding nucleic acid molecules. For instance, “Kappabodies” (Ill et al., Protein Eng, 10: 949-57 (1997)), “Minibodies”(Martin et al., EMBO J, 13: 5303-9 (1994)), “Diabodies” (Holliger etal., PNAS USA, 90: 6444-6448 (1993)), or “Janusins” (Traunecker et al.,EMBO J, 10:3655-3659 (1991) and Traunecker et al., “Janusin: newmolecular design for bispecific reagents,” Int J Cancer Suppl, 7:51-52(1992)) may be prepared using standard molecular biological techniquesfollowing the teachings of the specification.

In another aspect, chimeric and bispecific antibodies can be generated.A chimeric antibody may be made that comprises CDRs and frameworkregions from different antibodies. In a preferred embodiment, the CDRsof the chimeric antibody comprises all of the CDRs of the variableregion of a light chain or heavy chain of a human anti-MAdCAM antibody,while the framework regions are derived from one or more differentantibodies. In a more preferred embodiment, the CDRs of the chimericantibody comprise all of the CDRs of the variable regions of the lightchain and the heavy chain of a human anti-MAdCAM antibody. The frameworkregions may be from another species and may, in a preferred embodiment,be humanized. Alternatively, the framework regions may be from anotherhuman antibody.

A bispecific antibody can be generated that binds specifically to MAdCAMthrough one binding domain and to a second molecule through a secondbinding domain. The bispecific antibody can be produced throughrecombinant molecular biological techniques, or may be physicallyconjugated together. In addition, a single chain antibody containingmore than one VH and VL may be generated that binds specifically toMAdCAM and to another molecule. Such bispecific antibodies can begenerated using techniques that are well known for example, inconnection with (i) and (ii) see, e.g., Fanger et al., Immunol Methods4: 72-81 (1994) and Wright and Harris, supra. and in connection with(iii) see, e.g., Traunecker et al., Int. J. Cancer (Suppl.) 7: 51-52(1992). In a preferred embodiment, the bispecific antibody binds toMAdCAM and to another molecule expressed at high level on endothelialcells. In a more preferred embodiment, the other molecule is VCAM, ICAMor L-selectin.

In various embodiments, the modified antibodies described above areprepared using one or more of the variable regions or one or more CDRregions from one of the antibodies selected from 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In anotherembodiment, the modified antibodies are prepared using one or more ofthe variable regions or one or more CDR regions whose amino acidsequence is presented in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58,62, 64, 66 or 68 or whose nucleotide sequence is presented in SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 51, 53, 55, 57, 61, 63, 65 or 67.

Derivatized and Labeled Antibodies

An antibody or antibody portion of the invention can be derivatized orlinked to another molecule (e.g., another peptide or protein). Ingeneral, the antibodies or portions thereof are derivatized such thatthe MAdCAM binding is not affected adversely by the derivatization orlabeling. Accordingly, the antibodies and antibody portions of theinvention are intended to include both intact and modified forms of thehuman anti-MAdCAM antibodies described herein. For example, an antibodyor antibody portion of the invention can be functionally linked (bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody (e.g.,a bispecific antibody or a diabody), a detection agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate association of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antibody portion of theinvention may be derivatized include fluorescent compounds, includingfluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody may also be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody may be labeled with a magnetic agent, such as gadolinium. Anantibody may also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

An anti-MAdCAM antibody may also be labeled with a radiolabeled aminoacid. The radiolabel may be used for both diagnostic and therapeuticpurposes. For instance, the radiolabel may be used to detectMAdCAM-expressing tissues by x-ray or other diagnostic techniques.Further, the radiolabel may be used therapeutically as a toxin fordiseased tissue or MAdCAM expressing tumors. Examples of labels forpolypeptides include, but are not limited to, the followingradioisotopes or radionuclides—³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹TC, ¹¹¹In,¹²⁵I, ¹³¹I.

An anti-MAdCAM antibody may also be derivatized with a chemical groupsuch as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups may be useful to improve the biologicalcharacteristics of the antibody, e.g., to increase serum half-life or toincrease tissue binding. This methodology would also apply to anyantigen-binding fragments or versions of anti-MAdCAM antibodies.

Pharmaceutical Compositions and Kits

In a further aspect, the invention provides compositions comprising aninhibitory human anti-MAdCAM antibody and methods for treating subjectswith such compositions. In some embodiments, the subject of treatment ishuman. In other embodiments, the subject is a veterinary subject. Insome embodiments, the veterinary subject is a dog or a non-humanprimate.

Treatment may involve administration of one or more inhibitoryanti-MAdCAM monoclonal antibodies of the invention, or antigen-bindingfragments thereof, alone or with a pharmaceutically acceptable carrier.Inhibitory anti-MAdCAM antibodies of the invention and compositionscomprising them, can be administered in combination with one or moreother therapeutic, diagnostic or prophylactic agents. Additionaltherapeutic agents include anti-inflammatory or immunomodulatory agents.These agents include, but are not limited to, the topical and oralcorticosteroids such as prednisolone, methylprednisolone, NCX-1015 orbudesonide; the aminosalicylates such as mesalazine, olsalazine,balsalazide or NCX-456; the class of immunomodulators such asazathioprine, 6-mercaptopurine, methotrexate, cyclosporin, FK506, IL-10(Ilodecakin), IL-11 (Oprelevkin), IL-12, MIF/CD74 antagonists, CD40antagonists, such as TNX-100/5-D12, OX40L antagonists, GM-CSF,pimecrolimus or rapamycin; the class of anti-TNFα agents such asinfliximab, adalimumab, CDP-870, onercept, etanercept; the class ofanti-inflammatory agents, such as PDE-4 inhibitors (roflumilast, etc),TACE inhibitors (DPC-333, RDP-58, etc) and ICE inhibitors (VX-740, etc)as well as IL-2 receptor antagonists, such as daclizumab, the class ofselective adhesion molecule antagonists, such as natalizumab, MLN-02, oralicaforsen, classes of analgesic agents such as, but not limited to,COX-2 inhibitors, such as rofecoxib, valdecoxib, celecoxib, P/Q-typevoltage sensitize channel (α2δ) modulators, such as gabapentin andpregabalin, NK-1 receptor antagonists, cannabinoid receptor modulators,and delta opioid receptor agonists, as well as anti-neoplastic,anti-tumor, anti-angiogenic or chemotherapeutic agents Such additionalagents may be included in the same composition or administeredseparately. In some embodiments, one or more inhibitory anti-MAdCAMantibodies of the invention can be used as a vaccine or as adjuvants toa vaccine. In particular, because MAdCAM is expressed in lymphoidtissue, vaccine antigens can be advantageously targeted to lymphoidtissue by conjugating the antigen to an anti-MAdCAM antibody of theinvention.

As used herein, “pharmaceutically acceptable carrier” means any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption enhancing or delaying agents, and thelike that are physiologically compatible. Some examples ofpharmaceutically acceptable carriers are water, saline, phosphatebuffered saline, acetate buffer with sodium chloride, dextrose,glycerol, Polyethylene glycol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Additional examples ofpharmaceutically acceptable substances are surfactants, wetting agentsor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody.

The compositions of this invention may be in a variety of forms, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, lyophilized cake, dry powders, liposomesand suppositories. The preferred form depends on the intended mode ofadministration and therapeutic application. Typical preferredcompositions are in the form of injectable or infusible solutions, suchas compositions similar to those used for passive immunization ofhumans. The preferred mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular, intradermal).In a preferred embodiment, the antibody is administered by intravenousinfusion or injection. In another preferred embodiment, the antibody isadministered by intramuscular, intradermal or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, lyophilized cake, dry powder, microemulsion, dispersion,liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the anti-MAdCAM antibody in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by sterilization. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any any additionaldesired ingredient from a previously sterile solution thereof.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. The desiredcharacteristics of a solution can be maintained, for example, by the useof surfactants and the required particle size in the case of dispersionby the use of surfactants, phospholipids and polymers. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, for example,monostearate salts, polymeric materials, oils and gelatin.

The antibodies of the present invention can be administered by a varietyof methods known in the art, although for many therapeutic applications,the preferred route/mode of administration is subcutaneous,intramuscular, intradermal or intravenous infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

In certain embodiments, the antibody compositions may be prepared with acarrier that will protect the antibody against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems (J. R. Robinson, ed., MarcelDekker, Inc., New York (1978)).

In certain embodiments, an anti-MAdCAM antibody of the invention can beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the anti-MAdCAM antibodiescan be incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

The compositions of the invention may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of anantibody or antigen-binding portion of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount may be less thanthe therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a pre-determined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the anti-MAdCAM antibody or portion thereof and theparticular therapeutic or prophylactic effect to be achieved, and (b)the limitations inherent in the art of compounding such an antibody forthe treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg,more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. In someembodiments, a formulation contains 5 mg/mL of antibody in a buffer of20 mM sodium acetate, pH 5.5, 140 mM NaCl, and 0.2 mg/mL polysorbate 80.It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Another aspect of the present invention provides kits comprising ananti-MAdCAM antibody or antibody portion of the invention or acomposition comprising such an antibody. A kit may include, in additionto the antibody or composition, diagnostic or therapeutic agents. A kitcan also include instructions for use in a diagnostic or therapeuticmethod. In a preferred embodiment, the kit includes the antibody or acomposition comprising it and a diagnostic agent that can be used in amethod described below. In another preferred embodiment, the kitincludes the antibody or a composition comprising it and one or moretherapeutic agents that can be used in a method described below.

Gene Therapy

The nucleic acid molecules of the instant invention can be administeredto a patient in need thereof via gene therapy. The therapy may be eitherin vivo or ex vivo. In a preferred embodiment, nucleic acid moleculesencoding both a heavy chain and a light chain are administered to apatient. In a more preferred embodiment, the nucleic acid molecules areadministered such that they are stably integrated into chromosomes of Bcells because these cells are specialized for producing antibodies. In apreferred embodiment, precursor B cells are transfected or infected exvivo and re-transplanted into a patient in need thereof. In anotherembodiment, precursor B cells or other cells are infected in vivo usinga recombinant virus known to infect the cell type of interest. Typicalvectors used for gene therapy include liposomes, plasmids and viralvectors. Exemplary viral vectors are retroviruses, adenoviruses andadeno-associated viruses. After infection either in vivo or ex vivo,levels of antibody expression can be monitored by taking a sample fromthe treated patient and using any immunoassay known in the art ordiscussed herein.

In a preferred embodiment, the gene therapy method comprises the stepsof administering an isolated nucleic acid molecule encoding the heavychain or an antigen-binding portion thereof of an anti-MAdCAM antibodyand expressing the nucleic acid molecule. In another embodiment, thegene therapy method comprises the steps of administering an isolatednucleic acid molecule encoding the light chain or an antigen-bindingportion thereof of an anti-MAdCAM antibody and expressing the nucleicacid molecule. In a more preferred method, the gene therapy methodcomprises the steps of administering of an isolated nucleic acidmolecule encoding the heavy chain or an antigen-binding portion thereofand an isolated nucleic acid molecule encoding the light chain or theantigen-binding portion thereof of an anti-MAdCAM antibody of theinvention and expressing the nucleic acid molecules. The gene therapymethod may also comprise the step of administering anotheranti-inflammatory or immunomodulatory agent.

Diagnostic Methods of Use

The anti-MAdCAM antibodies may be used to detect MAdCAM in a biologicalsample in vitro or in vivo. The anti-MAdCAM antibodies may be used in aconventional immunoassay, including, without limitation, an ELISA, anRIA, FACS, tissue immunohistochemistry, Western blot orimmunoprecipitation. The anti-MAdCAM antibodies of the invention may beused to detect MAdCAM from humans. In another embodiment, theanti-MAdCAM antibodies may be used to detect MAdCAM from Old Worldprimates such as cynomolgus and rhesus monkeys, chimpanzees and apes.The invention provides a method for detecting MAdCAM in a biologicalsample comprising contacting a biological sample with an anti-MAdCAMantibody of the invention and detecting the antibody bound to MAdCAM. Inone embodiment, the anti-MAdCAM antibody is directly derivatized with adetectable label. In another embodiment, the anti-MAdCAM antibody (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the anti-MAdCAM antibody is labeled. As is well known toone of skill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the anti-MAdCAM antibody is a human IgG, then thesecondary antibody may be an anti-human-IgG. Other molecules that canbind to antibodies include, without limitation, Protein A and Protein G,both of which are available commercially, e.g., from Pierce Chemical Co.

Suitable labels for the antibody or secondary have been disclosed supra,and include various enzymes, prosthetic groups, fluorescent materials,luminescent materials, magnetic agents and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; an example of amagnetic agent includes gadolinium; and examples of suitable radioactivematerial include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, MAdCAM can be assayed in a biologicalsample by a competition immunoassay utilizing MAdCAM standards labeledwith a detectable substance and an unlabeled anti-MAdCAM antibody. Inthis assay, the biological sample, the labeled MAdCAM standards and theanti-MAdCAM antibody are combined and the amount of labeled MAdCAMstandard bound to the unlabeled antibody is determined. The amount ofMAdCAM in the biological sample is inversely proportional to the amountof labeled MAdCAM standard bound to the anti-MAdCAM antibody.

One may use the immunoassays disclosed above for a number of purposes.In one embodiment, the anti-MAdCAM antibodies may be used to detectMAdCAM in cells in cell culture. In a preferred embodiment, theanti-MAdCAM antibodies may be used to determine the level of cellsurface MAdCAM expression after treatment of the cells with variouscompounds. This method can be used to test compounds that may be used toactivate or inhibit MAdCAM. In this method, one sample of cells istreated with a test compound for a period of time while another sampleis left untreated, cell surface expression could then be determined byflow cytometry, immunohistochemistry, Western blot, ELISA or RIA. Inaddition, the immunoassays may be scaled up for high throughputscreening in order to test a large number of compounds for eitheractivation or inhibition of MAdCAM.

The anti-MAdCAM antibodies of the invention may also be used todetermine the levels of MAdCAM on a tissue or in cells derived from thetissue. In a preferred embodiment, the tissue is a diseased tissue. In amore preferred embodiment, the tissue is inflamed gastrointestinal tractor a biopsy thereof. In a preferred embodiment of the method, a tissueor a biopsy thereof is excised from a patient. The tissue or biopsy isthen used in an immunoassay to determine, e.g., MAdCAM levels, cellsurface levels of MAdCAM, or localization of MAdCAM by the methodsdiscussed above. The method can be used to determine if an inflamedtissue expresses MAdCAM at a high level.

The above-described diagnostic method can be used to determine whether atissue expresses high levels of MAdCAM, which may be indicative that thetissue will respond well to treatment with anti-MAdCAM antibody.Further, the diagnostic method may also be used to determine whethertreatment with anti-MAdCAM antibody (see below) is causing a tissue toexpress lower levels of MAdCAM and thus can be used to determine whetherthe treatment is successful.

The antibodies of the present invention may also be used in vivo tolocalize tissues and organs that express MAdCAM. In a preferredembodiment, the anti-MAdCAM antibodies can be used to localize inflamedtissue. The advantage of the anti-MAdCAM antibodies of the presentinvention is that they will not generate an immune response uponadministration. The method comprises the steps of administering ananti-MAdCAM antibody or a pharmaceutical composition thereof to apatient in need of such a diagnostic test and subjecting the patient toimaging analysis determine the location of the MAdCAM-expressingtissues. Imaging analysis is well known in the medical art, andincludes, without limitation, x-ray analysis, gamma scintigraphy,magnetic resonance imaging (MRI), positron emission tomography orcomputed tomography (CT). In another embodiment of the method, a biopsyis obtained from the patient to determine whether the tissue of interestexpresses MAdCAM rather than subjecting the patient to imaging analysis.In a preferred embodiment, the anti-MAdCAM antibodies may be labeledwith a detectable agent that can be imaged in a patient. For example,the antibody may be labeled with a contrast agent, such as barium, whichcan be used for x-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CT. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother embodiment, the anti-MAdCAM antibody will be unlabeled and willbe imaged by administering a second antibody or other molecule that isdetectable and that can bind the anti-MAdCAM antibody.

The anti-MAdCAM antibodies of the invention may also be used todetermine the levels of soluble MAdCAM present in donor blood, serum,plasma, or other biofluid, including, but not limited to, stool, urine,sputum or biopsy sample. In a preferred embodiment, the biofluid isplasma. The biofluid is then used in an immunoassay to determine levelsof soluble MAdCAM. Soluble MAdCAM could be a surrogate marker forongoing gastrointestinal inflammation and the method of detection couldbe used as a diagnostic marker to measure disease severity.

The above-described diagnostic method can be used to determine whetheran individual expresses high levels of soluble MAdCAM, which may beindicative that the individual will respond well to treatment with ananti-MAdCAM antibody. Further, the diagnostic method may also be used todetermine whether treatment with anti-MAdCAM antibody (see below) orother pharmaceutical agent of the disease is causing an individual toexpress lower levels of MAdCAM and thus can be used to determine whetherthe treatment is successful

Inhibition of α₄β₇/MAdCAM-Dependent Adhesion by Anti-MAdCAM Antibody:

In another embodiment, the invention provides an anti-MAdCAM antibodythat binds MAdCAM and inhibits the binding and adhesion of α₄β₇⁺-integrin bearing cells to MAdCAM or other cognate ligands, such asL-selectin, to MAdCAM. In a preferred embodiment, the MAdCAM is humanand is either a soluble form, or expressed on the surface of a cell. Inanother preferred embodiment, the anti-MAdCAM antibody is a humanantibody. In another embodiment, the antibody or portion thereofinhibits binding between α₄β₇ ⁺ and MAdCAM with an IC₅₀ value of no morethan 50 nM. In a preferred embodiment, the IC₅₀ value is no more than 5nM. In a more preferred embodiment, the IC₅₀ value is less than 5 nM. Ina more preferred embodiment, the IC₅₀ value is less than 0.05 μg/mL,0.04 μg/mL or 0.03 μg/mL. In another preferred embodiment the IC₅₀ valueis less than 0.5 μg/mL, 0.4 μg/mL or 0.3 μg/mL. The IC₅₀ value can bemeasured by any method known in the art. Typically, an IC₅₀ value can bemeasured by ELISA or adhesion assay. In a preferred embodiment, the IC₅₀value is measured by adhesion assay using either cells or tissue whichnatively express MAdCAM or cells or tissue which have been engineered toexpress MAdCAM.

Inhibition of Lymphocyte Recruitment to Gut-Associated Lymphoid Tissueby Anti-MAdCAM Antibodies

In another embodiment, the invention provides an anti-MAdCAM antibodythat binds natively expressed MAdCAM and inhibits the binding oflymphocytes to specialised gastrointestinal lymphoid tissue. In apreferred embodiment, the natively-expressed MAdCAM is human or primateMAdCAM and is either a soluble form, or expressed on the surface of acell. In another preferred embodiment, the anti-MAdCAM antibody is ahuman antibody. In another embodiment, the antibody or portion thereofinhibits the recruitment of gut-trophic α₄β₇ ⁺ lymphocytes to tissuesexpressing MAdCAM with an IC₅₀ value of no more than 5 mg/kg. In apreferred embodiment, the IC₅₀ value is no more than 1 mg/kg. In a morepreferred embodiment, the IC₅₀ value is less than 0.1 mg/kg. In oneembodiment, the IC₅₀ value can be determined by measuring the doseeffect relationship of recruitment of technetium-labeled peripheralblood lymphocytes to the gastrointestinal tract using gamma scintigraphyor single photon emission computed tomography. In an another embodiment,the IC₅₀ value can be determined by measuring the increase ingut-trophic α₄β₇ ⁺ lymphocytes, such as, but not limited to, CD4⁺ α₄β₇ ⁺memory T-cells, in the peripheral circulation using flow cytometry as afunction of the dose of anti-MAdCAM antibody.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

Example 1 Generation of Anti-MAdCAM Producing Hybridomas

Antibodies of the invention were prepared, assayed and selected inaccordance with the present Example

Primary Immunogen Preparation:

Two immunogens were prepared for immunisation of the XenoMouse™ mice:(i) a MAdCAM-IgG₁ Fc fusion protein and (ii) cell membranes preparedfrom cells stably transfected with MAdCAM.

(i) MAdCAM-IgG₁ Fc Fusion Protein

Expression Vector Construction:

An EcoRI/BglII cDNA fragment encoding the mature extracellular,immunoglobulin-like domain of MAdCAM was excised from a pINCY Incyteclone (3279276) and cloned into EcoRI/BamHI sites of the pIG1 vector(Simmons, D. L. (1993) in Cellular Interactions in Development: APractical Approach, ed. Hartley, D. A. (Oxford Univ. Press, Oxford), pp.93-127.)) to generate an in frame IgG₁ Fc fusion. The resulting insertwas excised with EcoRI/NotI and cloned into pCDNA3.1+ (Invitrogen). TheMAdCAM-IgG₁ Fc cDNA in the vector was sequence confirmed. The amino acidsequence of the MAdCAM-IgG₁ Fc fusion protein is shown below:

MAdCAM-IgG₁ Fc Fusion Protein:

(SEQ ID NO: 107) MDFGLALLLAGLLGLLLG QSLQVKPLQVEPPEPVVAVALGASRQLTCRLACADRGASVQWRGLDTSLGAVQSDTGRSVLTVRNASLSAAGTRVCVGSCGGRTFQHTVQLLVYAFPDQLTVSPAALVPGDPEVACTAHKVTPVDPNALSFSLLVGGQELEGAQALGPEVQEEEEEPQGDEDVLFRVTERWRLPPLGTPVPPALYCQATMRLPGLELSHRQAIPVLHSPTSPEPPDTTSPESPDTTSPESPDTTSQEPPDTTSQEPPDTTSQEPPDTTSPEPPDKTSPEPAPQQGSTHTPRSPGSTRTRRPEIQPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Underlined: signal peptideBold: MAdCAM extracellular domain

Recombinant Protein Expression/Purification:

CHO-DHFR cells were transfected with pCDNA3.1+ vector containingMAdCAM-IgG₁ Fc fusion protein cDNA and stable clones expressingMAdCAM-IgG₁ Fc fusion protein selected in Iscove's media containing 600μg/mL G418 and 100 ng/mL methotrexate. For protein expression, a hollowfibre bioreactor was seeded with stably expressing MAdCAM-IgG₁ Fc CHOcells in Iscove's media containing 10% low IgG fetal bovine serum(Gibco), non essential amino acids (Gibco), 2 mM glutamine (Gibco),sodium pyruvate (Gibco), 100 μg/mL G418 and 100 ng/mL methotrexate, andused to generate concentrated media supernatant. The MAdCAM-IgG₁ Fcfusion protein was purified from the harvested supernatant by affinitychromatography. Briefly, supernatant was applied to a HiTrap Protein GSepharose (5 mL, Pharmacia) column (2 mL/min), washed with 25 mM Tris pH8, 150 mM NaCl (5 column volumes) and eluted with 100 mM glycine pH 2.5(1 ml/min), immediately neutralising fractions to pH 7.5 with 1M Tris pH8. Fractions containing MAdCAM-IgG₁ Fc fusion protein were identified bySDS-PAGE, pooled together and applied to a Sephacryl S100 column(Pharmacia), pre-equilibrated with 35 mM BisTris pH 6.5, 150 mM NaCl.The gel filtration was performed at 0.35 ml/min, collecting a peak ofMAdCAM-IgG₁ Fc fusion protein in ca. 3×5 mL fractions. These sampleswere pooled and applied to a Resource Q (6 mL, Pharmacia) column,pre-equilibrated in 35 mM BisTris pH6.5. The column was washed with 5column volumes of 35 mM Bis Tris pH 6.5, 150 mM NaCl (6 ml/min) andMAdCAM-IgG₁ Fc fusion protein eluted into a 4-6 mL fraction with 35 mMBis Tris pH 6.5, 400 mM NaCl. At this stage the protein was 90% pure andmigrating as a single band at approximately 68 kD by SDS-PAGE. For useas an immunogen and all subsequent assays, the material was bufferexchanged into 25 mM HEPES pH 7.5, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 50%glycerol and stored as aliquots at −80° C.

(ii) Cell Membranes Stably Expressing MAdCAM

A SacI/NotI fragment comprising nucleotides 645-1222 of the publishedMAdCAM sequence (Shyjan A M, et al., J Immunol., 156, 2851-7 (1996)) wasPCR amplified from a colon cDNA library and cloned into SacI/NotI sitesof pIND-Hygro vector (Invitrogen). A SacI fragment, comprising theadditional 5′ coding sequence was sub-cloned into this construct frompCDNA3.1 MAdCAM-IgG₁ Fc, to generate the full length MAdCAM cDNA. AKpnI/NotI fragment containing the MAdCAM cDNA was then cloned intocorresponding sites in a pEF5FRTV5GWCAT vector (Invitrogen) andreplacing the CAT coding sequence. The cDNA insert was sequence verifiedand used in transfections to generate single stably expressing clones inFlpIn NIH 3T3 cells (Invitrogen) by Flp recombinase technology,according to the manufacturer's instructions. Stably expressing cloneswere selected by their ability to support the binding of a α₄β₇ ⁺ JYhuman B lymphoblastoid cell line (Chan B M, et al, J. Biol. Chem.,267:8366-70 (1992)), outlined below. Stable clones of CHO cellsexpressing MAdCAM were prepared in the same way, using FlpIn CHO cells(Invitrogen).

MAdCAM-expressing FlpIn NIH-3T3 cells were grown in Dulbecco's modifiedEagles Medium (Gibco), containing 2 mM L-glutamine, 10% Donor calf serum(Gibco) and 200 μg/mL Hygromycin B (Invitrogen) and expanded in rollerbottles. MAdCAM-expressing FlpIn CHO cells were grown in Ham'sF12/Dulbecco's modified Eagles Medium (Gibco), containing 2 mML-glutamine, 10% Donor calf serum (Gibco) and 350 μg/mL Hygromycin B(Invitrogen) and expanded in roller bottles. Cells were harvested by useof a non-enzymatic cell dissociation solution (Sigma) and scraping,washing in phosphate buffered saline by centrifugation. Cell membraneswere prepared from the cell pellet by two rounds of polytronhomogenization in 25 mM Bis Tris pH 8, 10 mM MgCl₂, 0.015% (w/v)aprotinin, 100 U/mL bacitracin and centrifugation. The final pellet wasresuspended in the same buffer, and 50×10⁶ cell equivalents aliquotedinto thick-walled eppendorfs and spun at >100,000 g to generate cellmembrane pellets for XenoMouse mice immunisations. Supernatant wasdecanted and membranes were stored in eppendorfs at −80° C. untilrequired. Confirmation of protein expression in the cell membranes wasdetermined by SDS-PAGE and Western blotting with a rabbit anti-peptideantibody raised against the N-terminal residues of MAdCAM([C]-KPLQVEPPEP).

Immunization and Hybridoma Generation:

Eight to ten week old XENOMOUSE™ mice were immunized intraperitoneallyor in their hind footpads with either the purified recombinantMAdCAM-IgG₁ Fc fusion protein (10 μg/dose/mouse), or cell membranesprepared from either stably expressing MAdCAM-CHO or NIH 3T3 cells(10×10⁶ cells/dose/mouse). This dose was repeated five to seven timesover a three to eight week period. Four days before fusion, the micereceived a final injection of the extracellular domain of human MAdCAMin PBS. Spleen and lymph node lymphocytes from immunized mice were fusedwith the non-secretory myeloma P3-X63-Ag8.653 cell line and weresubjected to HAT selection as previously described (Galfre and Milstein,Methods Enzymol. 73:3-46 (1981)). A panel of hybridomas all secretingMAdCAM specific human IgG₂κ and IgG₄κ antibodies were recovered andsub-cloned. Twelve hybridoma sub-clones, 1.7.2, 1.8.2, 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2,producing monoclonal antibodies specific for MAdCAM were recovered anddetected with assays described below. The parental lines 1.7, 1.8, 6.14,6.22, 6.34, 6.67, 6.73, 6.77, 7.16, 7.20, 7.26 and 9.8, from which thesub-clone hybridoma lines, 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1,6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2, were derived all hadanti-MAdCAM activity.

ELISA Assays:

Detection of antigen-specific antibodies in mouse serum and hybridomasupernatant was determined by ELISA as described (Coligan et al., Unit2.1 “Enzyme-linked immunosorbent assays,” in Current Protocols inImmunology (1994)) using MAdCAM-IgG₁ Fc fusion protein to capture theantibodies. For animals that were immunised with MAdCAM-IgG₁ Fc fusionprotein, antibodies were screened for non-specific reactivity againsthuman IgG₁ and for the ability to bind to FlpIn CHO MAdCAM cells by flowcytometry.

In a preferred ELISA assay, the following techniques are used:

ELISA plates were coated overnight at 4° C. with 100 μL/well ofMAdCAM-IgG₁ Fc fusion (4.5 μg/mL) in plate containing buffer (100 mMsodium carbonate/bicarbonate buffer pH 9.6). After incubation, coatingbuffer was removed and the plate blocked with 200 μL/well blockingbuffer (5% BSA, 0.1% Tween 20, in phosphate buffered saline) andincubated at room temperature for 1 hour. Blocking buffer was removedand 50 μL/well of hybridoma supernatant or other serum or supernatant(e.g., positive control) added for 2 hours at room temperature. Afterincubation the plate was washed with PBS (3×100 μL/well) and the bindingof the hybridoma mAb detected with HRP-conjugated secondary antibodies(i.e. 1:1000 mouse anti-human IgG₂-HRP (SB Cat. No. 9060-05) for IgG₂antibodies or 1:1000 mouse anti-human IgG₄-HRP (Zymed Cat. No. 3840) forIgG₄ antibodies) diluted in PBS. The plates were incubated at roomtemperature for 1 hour, washed in PBS (3×100 μL/well) and finallydeveloped with 100 μL, OPD (o-phenylenediamine (DAKO S2405)+5 μL 30%H₂O₂/12 mL). The plates were allowed to develop 10-20 mins, stopping thereaction with 100 μL, 2M H₂SO₄. The plates were read at 490 nm.

Adhesion Assays:

Antibodies that demonstrated binding to MAdCAM-IgG₁ Fc fusion protein byELISA, were assessed for antagonist activity in an adhesion assays withα₄β₇ ⁺ JY cells and either (i) MAdCAM-IgG₁ Fc fusion protein or (ii)MAdCAM-CHO cells.

(i) MAdCAM-IgG₁ Fc Fusion Assay

100 μL of a 4.5 μg/mL solution of purified MAdCAM-IgG₁ Fc fusion proteinin Dulbecco's PBS was adsorbed to 96 well Black Microfluor “B” u-bottom(Dynex #7805) plates overnight at 4° C. The MAdCAM coated plates werethen inverted and excess liquid blotted off, prior to blocking at 37° C.for at least 1 hour in 10% BSA/PBS. During this time cultured JY cellswere counted using tryptan blue exclusion (should be approximately 8×10⁵cells/mL) and 20×10⁶ cells/assay plate pipetted into a 50 mL centrifugetube. JY cells were cultured in RPMI1640 media (Gibco), containing 2 mML-glutamine and 10% heat-inactivated fetal bovine serum (LifeTechnologies #10108-165) and seeded at 1-2×10⁵/mL every 2-3 days toprevent the culture from differentiating. The cells were washed twicewith RPMI 1640 media (Gibco) containing 2 mM L-glutamine (Gibco) bycentrifugation (240 g), resuspending the final cell pellet at 2×10⁶cells/mL in RPMI 1640 for Calcein AM loading. Calcein AM (MolecularProbes #C-3099) was added to the cells as a 1:200 dilution in DMSO (ca.final concentration 5 μM) and the cells protected from light during thecourse of the incubation (37° C. for 30 min). During this cellincubation step the antibodies to be tested, were diluted as follows:for single dose testing, the antibodies were made up to 3 μg/mL (1 μg/mLfinal) in 0.1 mg/mL BSA (Sigma#A3059) in PBS; for full IC₅₀ curves, theantibodies were diluted in 0.1 mg/mL BSA/PBS, with 3 μg/mL (1 μg/mLfinal) being the top concentration, then doubling dilutions (1:2 ratio)across the plate. The final well of the row was used for determiningtotal binding, so 0.1 mg/ml BSA in PBS was used.

After blocking, the plate contents were flicked out and 50 μL ofantibodies/controls were added to each well and the plate incubated at37° C. for 20 mM. During this time, Calcein-loaded JY cells were washedonce with RPMI 1640 media containing 10% fetal bovine serum and oncewith 1 mg/mL BSA/PBS by centrifugation, resuspending the final cellpellet to 1×10⁶/mL in 1 mg/mL BSA/PBS. 100 μL of cells were added toeach well of the U bottomed plate, the plate sealed, briefly centrifuged(1000 rpm for 2 mM) and the plate then incubated at 37° C. for 45 mM. Atthe end of this time, the plates were washed with a Skatron plate washerand fluorescence measured using a Wallac Victor² 1420 Multilabel Reader(excitation λ 485 nm, emission λ 535 nm count from top, 8 mm from bottomof plate, for 0.1 sec with normal emission aperture). For each antibodyconcentration, percent adhesion was expressed as a percentage of maximalfluorescence response in the absence of any antibody minus fluorescenceassociated with non-specific binding. The IC₅₀ value is defined as theanti-MAdCAM antibody concentration at which the adhesion response isdecreased to 50% of the response in the absence of anti-MAdCAM antibody.Antibodies that were able to inhibit the binding of JY cells toMAdCAM-IgG₁ Fc fusion with an IC₅₀ value <0.1 μg/mL, were considered tohave potent antagonist activity and were progressed to the MAdCAM-CHOadhesion assay. All twelve of the tested Abs showed potent antagonistactivity (Table 3). Monoclonal antibodies 1.7.2, 1.8.2, 7.16.6, 7.20.5and 7.26.4 were derived from IgG₂K lineages, and monoclonal antibodies6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1 and 9.8.2 were derivedfrom IgG₄K lineages.

(ii) MAdCAM-CHO Cell Adhesion Assay.

JY cells were cultured as above. MAdCAM-expressing CHO cells weregenerated with the pEF5FRT MAdCAM cDNA construct and using the Flprecombinase technology (Invitrogen) as described above. Single stableclones of MAdCAM-expressing CHO cells were selected based on theirability to support the adhesion of JY cells and the binding, by flowcytometry, of the rabbit anti-peptide antibody, raised against theN-terminus of MAdCAM and described above. MAdCAM-expressing CHO cellswere cultured in a DMEM/F12 media (Gibco #21331-020) containing 2 mML-glutamine, 10% fetal bovine serum (Gibco) and 350 μg/mL Hygromycin B(Invitrogen), splitting 1:5 every 2/3 days. For the adhesion assay,MAdCAM-expressing CHO cells were seeded at 4×10⁴ cells/well in 96 wellblack plates—clear bottom (Costar #3904) in 200 μL culture medium andcultured overnight at 37° C./5% CO₂.

The following day, hybridoma supernatant or purified monoclonal antibodywas diluted from a starting concentration of 30 μg/mL (equivalent to afinal concentration of 10 μg/mL) in 1 mg/mL BSA/PBS, as described above.For the MAdCAM CHO plates, the plate contents were flicked out and 50 μLof antibodies/controls were added to each well and the plate incubatedat 37° C. for 20 min. The final well of the row was used for determiningtotal binding, so 0.1 mg/mL BSA in PBS was used. Calcein AM-loaded JYcells, to a final concentration of 1×10⁶/mL in 1 mg/mL BSA/PBS, wereprepared as above, then 100 μL added to the plate after the 20 minincubation period with the antibody. The plate was then incubated at 37°C. for 45 min, then washed on a Tecan plate washer (PW 384) andfluorescence measured using the Wallac plate reader as described above.For each antibody concentration, percent adhesion was expressed as apercentage of maximal fluorescence response in the absence of anyantibody minus fluorescence associated with non-specific binding.Antibodies that were able to inhibit the binding of JY cells to MAdCAMCHO cells with an IC₅₀ value <1 μg/mL were considered to have potentantagonist activity. As before, the IC₅₀ value is defined as theanti-MAdCAM antibody concentration at which the adhesion response haddecreased to 50% of the response in the absence of anti-MAdCAM antibody.The IC₅₀ potencies for 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1,6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 in this assay aredescribed below in Table 3.

TABLE 3 IC₅₀ values of exemplified anti-MAdCAM antibodies MAdCAM IgG1MAdCAM FlpIn Fc fusion CHO Assay Mean IC50 Mean IC50 Clone (μg/mL) n(μg/mL) n IgG2 1.7.2 0.030 ± 0.011 6 0.052 ± 0.280 9 1.8.2 0.027 ± 0.0114 0.424 ± 0.107 8 7.16.6 0.019 ± 0.009 7 0.389 ± 0.093 16 7.20.5 0.025 ±0.027 7 0.387 ± 0.202 9 7.26.4 0.021 ± 0.040 4 0.574 ± 0.099 15 IgG46.14.2 0.011 ± 0.005 4 0.291 ± 0.096 6 6.22.2 0.018 ± 0.011 4 0.573 ±0.168 7 6.34.2 0.013 ± 0.008 4 0.285 ± 0.073 7 6.67.1 0.013 ± 0.070 40.298 ± 0.115 8 6.73.2 0.020 ± 0.010 4 0.369 ± 0.103 8 6.77.1 0.022 ±0.004 4 0.520 ± 0.100 4 9.8.2 0.020 ± 0.050 4 0.440 ± 0.342 8

To measure the antagonist potency of anti-MAdCAM mAbs in flow-basedassays, under sheer stress conditions that are designed to mimic themicrovascular environment on the high endothelial venules which servethe gut associated lymphoid tissue, CHO cells expressing MAdCAM wereplated in glass microslides (50×4 mm) and allowed to adhere to form aconfluent monolayer (ca. 2.5×10⁵ cells). The cells were then incubatedwith affinity-purified mAb over a range of concentrations (0.1-10 μg/mL)for 20 mins at 37° C., before being connected to the flow assay system.An isotype matched IgG₂ or IgG₄ mAb (10 μg/mL) was used as a negativecontrol. Normal donor peripheral blood lymphocytes (PBLs) were perfusedover the cell monolayer at a constant shear stress of 0.05 Pa.Experiments were videoed and total adhesion of lymphocytes (rolling+firmadhesion) was calculated. All of the tested monoclonal antibodies wereshown to be potent antagonists under the conditions described.

(iii) Stamper-Woodruff Assays

To visualise MAdCAM vessels, biotinylated anti-MAdCAM mAb was generatedon 1-2 mg of affinity-purified protein, using a 20 molar excess ofbiotin-NHS (Pierce) in phosphate buffer saline, according tomanufacturer's instructions. The reaction was allowed to sit at roomtemperature (30 min), and desalted with a PD-10 (Pharmacia) column andthe protein concentration determined.

Normal liver lymph node was removed from a donor organ, snap-frozen inliquid nitrogen and stored at −70° C. until use. 10 μm cryostat sectionswere cut, air-dried on poly-L lysine coated slides, and fixed in acetoneprior to the assay. Sections were blocked using an avidin-biotinblocking system (DAKO), and then incubated with biotinylated anti-MAdCAMmAb over a range of concentrations (1-50 μg/mL) at room temperature (2hrs). An isotype matched IgG₂ or IgG₄ mAb (50 μg/mL) was used as anegative control and a blocking anti-β₇ antibody (50 μg/mL) as apositive control.

Peripheral blood lymphocytes, taken from normal donors, were labeledwith a mouse anti-human CD2 mAb (DAKO) to allow subsequent visualisationof adherent cells. 5×10⁵ PBLs were added to each lymph node section andincubated for 30 mins before being gently rinsed off to avoid detachmentof adherent cells. Sections were then re-fixed in acetone, andre-incubated with biotinylated anti-MAdCAM mAb (10 μg/mL), followed bybiotinylated goat-anti-mouse mAb (to recognise CD2 labeled PBLs andunstained MAdCAM vessels) and then streptABcomplex/HRP (DAKO). FinallyMAdCAM vessels & CD2 labeled PBLs were visualised by addition of DABsubstrate (DAKO) to the sections, with a brown reaction product showingareas of positive staining. Lymphocyte adhesion was quantified bycounting the number of lymphocytes adhering to 50 MAdCAM-1⁺ vessels ofportal tracts, veins or sinusoids. Data, expressed as mean values, werethen normalised to percent adhesion, using the adhesion of PBLs in theabsence of any antibody taken as 100%. The data were compiled on thebasis of n=3 different PBL donors and for different liver lymph nodedonors. Representative data for biotinylated purified monoclonalantibodies 1.7.2 and 7.16.6 are depicted in FIG. 4 compared to ablocking anti-β₇ antibody control.

Selectivity Assays:

VCAM and fibronectin are close structural and sequence homologues toMAdCAM. Affinity-purified anti-MAdCAM mAbs were assessed forMAdCAM-specificity by determining their ability to block the binding ofα₄β₁ ⁺/α₅β₁ ⁺ Jurkat T-cells (ATCC) to their cognate cell adhesionmolecule. 100 μL of a 4.5 μg/mL solution of Fibronectin cell bindingfragment (110 Kd, Europa Bioproducts Ltd, Cat. No. UBF4215-18) or VCAM(Panvera) in Dulbecco's PBS was adsorbed to 96 well Black Microfluor “B”u-bottom (Dynex #7805) plates overnight at 4° C. The coated plates werethen inverted and excess liquid blotted off, prior to blocking at 37° C.for at least 1 hour in 10% BSA/PBS. During this time cultured Jurkat Tcells were counted using tryptan blue exclusion and loaded with CalceinAM dye as previously described for JY cells above. The antibodies to betested, were diluted from a top concentration of 10 μg/mL in 0.1 mg/mlBSA in PBS. The final well of the row was used for determining totalbinding, so 0.1 mg/ml BSA in PBS was used. Echistatin (Bachem, Cat. No.H-9010) prepared in PBS was used at a top concentration of 100 nM toblock the α₅β₁/Fibronectin interaction. An anti-CD106 mAb (Clone51-10C9, BD Pharmingen Cat. No. 555645) at a top concentration of 1μg/mL was used to block the α₄β₁/VCAM interaction.

After blocking, the plate contents were flicked out and 50 μL ofantibodies/controls were added to each well and the plate incubated at37° C. for 20 min. Calcein-loaded Jurkat T cells were washed once asbefore, resuspending the final cell pellet to 1×10⁶/mL in 1 mg/mLBSA/PBS. 100 μL of cells were added to each well of the U bottomedplate, the plate sealed, briefly centrifuged (1000 rpm for 2 min) andthe plate then incubated at 37° C. for 45 min. At the end of this time,the plates were washed with a Skatron plate washer and fluorescencemeasured using a Wallac Victor² 1420 Multilabel Reader (excitation λ485nm, emission λ535 nm count from top, 8 mm from bottom of plate, for 0.1sec with normal emission aperture). For each antibody, the degree ofinhibition is expressed below pictorially, in Table 4 (− negligibleinhibition of adhesion, *** complete inhibition of adhesion). All mAbsexemplified are potent and selective anti-MAdCAM antagonists,demonstrating substantially greater than 100 fold selectivity for MAdCAMover VCAM and fibronectin.

TABLE 4 Comparative selectivity of anti-MAdCAM antibody for MAdCAM overother cell adhesion molecules, Fibronectin and VCAM Inhibition inInhibition in Inhibition in α5β1/Fibronectin α4β1/VCAM α4β7/MAdCAM Cloneassay (10 μg/mL) assay (10 μg/mL) assay (0.1 μg/mL) IgG2 1.7.2 — — ***1.8.2 — — *** 7.16.6 — — *** 7.20.5 — — *** 7.26.4 — — *** IgG4 6.14.2 —— *** 6.22.2 — — *** 6.34.2 — — *** 6.67.1 — — *** 6.73.2 — — *** 6.77.1— — *** 9.8.2 — — *** — —

Hybridomas were deposited in the European Collection of Cell Cultures(ECACC), H.P.A at CAMR, Porton Down, Salisbury, Wiltshire SP4 0JG on 9Sep. 2003 with the following deposit numbers:

Hybridoma Deposit No. 1.7.2 03090901 1.8.2 03090902 6.14.2 030909036.22.2 03090904 6.34.2 03090905 6.67.1 03090906 6.73.2 03090907 6.77.103090908 7.16.6 03090909 7.20.5 03090910 7.26.4 03090911 9.8.2 03090912

Example II Determination of Affinity Constants (K_(d)) of Fully HumanAnti-MAdCAM Monoclonal Antibodies by BIAcore

We performed affinity measures of purified antibodies by surface plasmonresonance using the BIAcore 3000 instrument, following themanufacturer's protocols.

Protocol 1

To perform kinetic analyses, a high density mouse anti-human (IgG₂ andIgG₄) antibody surface over a CMS BIAcore sensor chip was prepared usingroutine amine coupling. Hybridoma supernatants were diluted 10, 5,2-fold in HBS-P (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Surfactant P20)running buffer containing 100 μg/mL BSA and 10 mg/mLcarboxymethyldextran or used neat. Each mAb was captured onto a separatesurface using a 1 mM contact time and a 5 mM wash for stabilization ofthe mAb baseline. MAdCAM-IgG₁ Fc (141 nM) fusion protein was theninjected at over all surfaces for one minute, followed by a 3 mMdissociation. The data were normalized for the amount of antibodycaptured on each surface and evaluated with global fit Langmuir 1:1,using baseline drift models available on the BIAevaluation softwareprovided by BIAcore.

Protocol 2

Affinity-purified mAb were immobilized onto the dextran layer of a CMSbiosensor chip using amine coupling. Chips were prepared using pH 4.5acetate buffer as the immobilization buffer and protein densities of2.5-5.5 kRU were achieved. Samples of MAdCAM-IgG₁ Fc fusion protein inrunning buffer were prepared at concentrations ranging from 0.2-55 nM (a0 nM solution comprising running buffer alone was included as a zeroreference). Samples were randomized and injected in duplicate for 3 mMeach across 4 flow cells using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl,3 mM EDTA, 0.005% Surfactant P20) as running buffer. A flow rate of 100μL/min was used to minimize mass transport limitations. Dissociation ofMAdCAM-IgG₁ Fc fusion protein was monitored for 180 mins, the surfaceregenerated by a 6 sec injection of 25 mM H₃PO₄ (50 μL/min), or 10 mM(6.22.2), 20 mM (6.67.1, 6.73.2, 6.77.1) to 25 mM (6.34.2) and 45 mMNaOH (6.14.2) and the data analysed using the BIAevaluation (v3.1)software package.

Table 5 lists affinity measurements for representative anti-MAdCAMantibodies of the present invention:

TABLE 5 Determination of affinity constant, K_(d), by surface plasmonresonance (BIAcore) Protocol 1 Protocol 2 K_(on) K_(off) K_(D) K_(on)K_(off) K_(D) CLONE (1/Ms) (1/s) (pM) (1/MS) (1/s) (pM) IgG2 1.7.2 2.4 ×10⁵  1 × 10⁻⁵ 42 5.5 × 10³ 1.3 × 10⁻⁷ 23.6 1.8.2 2.9 × 10⁵  1 × 10⁻⁵ 351.8 × 10⁵ 2.3 × 10⁻⁵ 128 7.16.6 1.5 × 10⁶ 2.2 × 10⁻⁶ 1.5 2.9 × 10⁵ 1.4 ×10⁻⁶ 4.8 7.20.5 4.5 × 10⁵ 1.9 × 10⁻⁵ 42.2 1.6 × 10⁵ 1.2 × 10⁻⁵ 75 7.26.49.6 × 10⁵ 2.6 × 10⁻⁴ 271 1.5 × 10⁵ 1.2 × 10⁻⁵ 80 IgG4 6.14.2 1.3 × 10⁵ 1 × 10⁻⁵ 7.7  5 × 10⁵  <5 × 10⁻⁶ <10 6.22.2 1.5 × 10⁶ 1.4 × 10⁻⁵ 9.32.3 × 10⁵ 8.7 × 10⁻⁷ 3.8 6.34.2 1.2 × 10⁶ 1.9 × 10⁻⁵ 15.8 3.3 × 10⁵  <5× 10⁻⁶ <15 6.67.1 5.9 × 10⁵  1 × 10⁻⁵ 17 2.4 × 10⁵  <5 × 10⁻⁶ <20 6.73.21.4 × 10⁵ 1.3 × 10⁻⁴ 93 6.77.1 1.5 × 10⁵  1 × 10⁻⁵ 6.7 9.8.2 2.3 × 10⁶2.3 × 10⁻⁴ 100 4.4 × 10⁵ 1.4 × 10⁻⁵ 32.5

The kinetic analyses indicate that the antibodies prepared in accordancewith the invention possess high affinities and strong binding constantsfor the extracellular domain of MAdCAM.

Example III Identification of Epitope Selectivity and SpeciesCross-Reactivity of Anti-MAdCAM mAbs

Antibodies recognize surface-exposed epitopes on antigens as regions oflinear (primary) sequence or structural (secondary) sequence. Luminexepitope binning, BIAcore binning and species immunohistochemicalanalysis were used in concert, in order to define the functional epitopelandscape of the anti-MAdCAM antibodies.

Luminex-Based Epitope Binning:

MxhIgG 2,3.4-conjugated beads (Calbiochem Ml 1427) were coupled to theprimary unknown anti-MAdCAM antibody. We added 150 μL of primary unknownantibody dilution (0.1 μg/mL diluted in hybridoma medium) to the well ofa 96-well tissue culture plate. The bead stock was gently vortexed anddiluted in supernatant to a concentration of 0.5×10⁵ beads/mL. The beadswere incubated in the supernatant on a shaker overnight in the dark at4° C.

Each well of a 96-well microtiter filter plate (Millipore # MABVN1250)was pre-wetted by adding 200 μL wash buffer (PBS containing 0.05%Tween20) and removed by aspiration. Next, 50 μL/well of the 0.5×10⁵beads/mL stock was added to the filter plate, and the wells washed withwash buffer (2×100 μL/well). 60 μL/well of MAdCAM-IgG₁ Fc antigendiluted in hybridoma medium (0.1 μg/mL) was added. The plates werecovered and incubated at room temperature with gentle shaking for onehour. The wells were washed twice by addition of 100 μL/well wash bufferfollowed by aspiration. Next, we added 60 μL/well of secondary unknownanti-MAdCAM antibody diluted in hybridoma medium (0.1 μg/mL). The plateswere shaken at room temperature in the dark for two hours. Next, thewells were washed twice by addition of 100 μL/well wash buffer followedby aspiration. Next, 60 μL/well of biotinylated MxhIgG 2,3,4 (0.5 μg/mL)was added. The plates were shaken at room temperature in the dark forone hour. The wells were washed twice by addition of 100 μL/well washbuffer followed by aspiration. To each well, 60 μL of 1 μg/mL MxhIgG2,3,4 Streptavidin-PE (Pharmacia #554061) diluted in hydridoma mediumwas added. The plates were shaken at room temperature in the dark fortwenty minutes. The wells were washed twice by addition of 100 μL/wellwash buffer followed by aspiration. Next, each well was resuspended in80 μL blocking buffer (PBS with 0.5% bovine serum albumin, 0.1% TWEENand 0.01% Thimerosal) carefully pipetted up and down to resuspend thebeads.

Using Luminex 100 and its accompanying software (Luminex® Corporation)the plates were read to determine luminescence readings. Based on theluminescence data obtained for the various anti-MAdCAM antibodiestested, the anti-MAdCAM antibodies were grouped according to theirbinding specificities. The anti-MAdCAM antibodies that were tested fallinto a series of epitope bins, represented in Table 8.

BIAcore Binning:

In a similar method to that described above, BIAcore can also be used todetermine the epitope exclusivity of the anti-MAdCAM antibodiesexemplified by this invention. Nine anti-MAdCAM antibody clones, 6.22.2,6.34.2, 6.67.1, 6.77.1, 7.20.5, 9.8.2, 1.7.2, 7.26.4 and 7.16.6, wereimmobilized onto the dextran layer of separate flow cells of a CMSbiosensor chip using amine coupling. The immobilization buffer waseither 10 mM acetate buffer pH 4.5 (clones 6.22.2, 6.34.2, 7.20.5,9.8.2, 1.7.2, 7.26.4 and 7.16.6) or 10 mM acetate buffer pH 5.5 (clones6.67.1 and 6.77.1). A protein density of approximately 3750 RU wasachieved in all cases. Deactivation of unreacted N-hydroxysuccinimideesters was performed using 1 M ethanolamine hydrochloride, pH 8.5.

MAdCAM-IgG₁ Fc fusion protein was diluted to a concentration of 1.5μg/mL (approximately 25 nM) in HBS-EP running buffer (0.01 M HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Polysorbate 20). It was theninjected across the first flow cell, in a volume of 50 μL at a rate of 5μL/min. After the injection was complete, the first antibody probe wasadded to the same flow cell. All test antibodies were diluted to aconcentration of approximately 20 μg/mL in HBS-EP, and also injected ina volume of 50 μL at a flow rate of 5 μL/min. When no binding of thetest antibody was observed, the next test clone was injected immediatelyafterwards. When binding did occur, the sensor surface was regeneratedto remove both the MAdCAM-IgG₁ Fc fusion protein and the test antibody.A variety of regeneration solutions were used depending upon theimmobilized antibody and the test antibody present. A summary of theregeneration conditions used is depicted in Table 6.

TABLE 6 Summary of regeneration conditions used to perform BIAcoreepiope mapping Immobilised Antibody probe Injection antibody to beremoved Regeneration solution volume 7.16.6 6.22.2 40 mM Phosphoric Acid20 μL 6.34.2 40 mM Phosphoric Acid 40 μL 7.20.5 40 mM Phosphoric Acid 20μL 6.77.1 9.8.2 40 mM Phosphoric Acid 10 μL 1.7.2 40 mM Phosphoric Acid 5 μL 7.16.6 40 mM Phosphoric Acid 10 μL 1.7.2 6.77.1 25 mM PhosphoricAcid  5 μL 9.8.2 25 mM Phosphoric Acid  5 μL 7.20.5 25 mM PhosphoricAcid  5 μL 6.22.2 25 mM Phosphoric Acid  5 μL 6.34.2 25 mM SodiumHydroxide  5 μL 6.67.1 25 mM Sodium Hydroxide  5 μL 6.22.2 9.8.2 25 mMSodium Hydroxide 20 μL 7.26.4 25 mM Sodium Hydroxide  5 μL 6.34.2 9.8.225 mM Sodium Hydroxide 70 μL 1.7.2 40 mM Sodium Hydroxide  5 μL 7.26.440 mM Sodium Hydroxide  5 μL 6.67.1 9.8.2 40 mM Sodium Hydroxide  5 μL1.7.2 40 mM Sodium Hydroxide  5 μL 7.20.5 9.8.2 25 mM Phosphoric Acid  5μL 1.7.2 25 mM Phosphoric Acid  5 μL 7.26.4 25 mM Phosphoric Acid  5 μL7.26.4 9.8.2 40 mM Sodium Hydroxide 20 μL 6.22.2 75 mM Phosphoric Acid20 μL 7.20.5 75 mM Phosphoric Acid 20 μL 7.16.6 75 mM Phosphoric Acid 20μL 9.8.2 9.8.2 25 mM Phosphoric Acid 15 μL 6.22.2 25 mM Phosphoric Acid10 μL 7.20.5 25 mM Phosphoric Acid 20 μL 7.16.6 25 mM Phosphoric Acid 10μL (Flow rate was 50 μL/min during all regeneration procedures)

After regeneration, MAdCAM-IgG₁ Fc fusion protein was bound again andfurther test antibodies were injected. These procedures were carried outuntil the entire panel of clones had been injected over the surface ofthe immobilised antibody, with bound MAdCAM-IgG₁ Fc fusion protein. Anew flow cell with a different immobilised antibody and bound MAdCAM wasthen used for probing with the nine test clones. Anti-MAdCAM antibodies1.7.2 and 1.8.2 were expected to recognise the same MAdCAM epitope,based on the close primary amino acid sequence homology of their heavyand kappa light chains, SEQ ID NOS: 2, 4, 6, 8 respectively.Accordingly, only 1.7.2 was assessed though the BIAcore response matrix.Antibodies 6.14.2 and 6.73.2 were omitted from this analysis, but allother combinations of anti-MAdCAM antibody pairs were tested in thisway. An arbitrary level of 100 RU was chosen as the threshold betweenbinding/non-binding and a response matrix, (Table 7), was created basedon whether binding was observed.

TABLE 7 BIAcore epitope binning response matrix Immobilised Secondaryantibody antibody 6.22.2 6.34.2 6.67.1 6.77.1 7.20.5 9.8.2 1.7.2 7.26.47.16.6 6.22.2 — — — — — x x x x 6.34.2 — — — — — x x x x 6.67.1 — — — —— x x — — 6.77.1 — — — — — x x — x 7.20.5 — — — — — x x x x 9.8.2 x x xx x x — — x 1.7.2 x x x x x x — — x 7.26.4 x x — — x x — — x 7.16.6 x x— — x — — — x Response matrix for all combinations of antibody pairs. —indicates no binding of the antibody probe, x indicates binding wasobserved (above a chosen threshold level of 100 RU).

The matrix diagonal in Table 7 (shaded grey) holds the binding data foridentical probe pairs. In all instances, except for the two clones7.16.6 and 9.8.2, the antibodies were self-blocking. Antibodies 7.16.6and 9.8.2 do not cross compete. The lack of self-blocking could be dueto a mAb-induced conformational change in the fusion protein thatpermits additional binding of the mAb to a second site on MAdCAM-IgFc.

Grouping the clones that show the same reactivity pattern gives rise toat least six different epitope bins, as shown in the graphicalrepresentation, FIG. 5).

Further precise identification of the MAdCAM epitope sequences withwhich an anti-MAdCAM antibody interacts can be determined by any of anumber of methods, including, but not limited to, Western analysis ofspotted peptide library arrays (Reineke et al., Curr. Topics inMicrobiol. and Immunol 243: 23-36 (1999), M. Famulok, E-L Winnacker, C-HWong eds., Springer-Verlag, Berlin), phage or bacterial flagellin/fliCexpression library display, or simple MALDI-TOF analysis of boundprotein fragments following limited proteolysis.

Immunohistochemical Assays:

OCT or sucrose-embedded frozen tissue specimens of ileum (Peyer'spatches), mesenteric lymph node, spleen, stomach, duodenum, jejunum andcolon were used as a positive staining controls for the anti-MAdCAMmAbs. For staining human sections with human IgG₂ mAbs, biotinylatedderivatives of the anti-MAdCAM mAbs were generated. 10 μm frozen tissuesections were cut onto poly L-lysine coated slides, placed directly into100% acetone 4° C. (10 min), then 3% hydrogen peroxide in methanol (10min), washing between steps with PBS. The slides were blocked withBiotin Blocking System (DAKO Cat. No. X0590), prior to incubation withthe primary antibody (1:100-1:1000) in PBS (1 hr), washed with PBS-Tween20 (0.05%) and then binding developed with HRP-Streptavidin (BDBioscience Cat. No. 550946, 30 min) and DAB substrate (Sigma Cat. No.D5905). For IgG₄ mAbs, an HRP-conjugated, mouse anti-human IgG₄ (ZymedCat. No. 3840) secondary was used. The slides were counterstained withMayer's Haemalum (1 min), washed and then mounted in DPX.

Binding affinity was compared for a number of species (mouse, rat,rabbit, dog, pig, cynomolgus and human tissue). There was no reactivityfor rat, rabbit and pig tissue by immunohistochemistry and nocross-reactivity of the anti-MAdCAM antibodies for recombinant mouseMAdCAM, when analyzed by

ELISA. The data for human, cynomolgus and dog tissue are presented intable form, Table 8 below:

TABLE 8 Pattern of cross reactivity of anti-MAdCAM antibodies to MAdCAMspecies orthologues IHC cross-reactivity Luminex human cyno marmoset dogCLONE BIN ileum ileum ileum ileum IgG2 1.7.2 3a Binding No Binding NoBinding No Binding 1.8.2 3a Binding No Binding No Binding No Binding7.16.6 3b Binding Binding No Binding No Binding No Binding 7.20.5 2bBinding Binding N.D. No Binding 7.26.4 3b Binding Binding N.D. NoBinding IgG4 6.14.2 2 Binding Binding N.D. Binding 6.22.2 2 BindingBinding N.D. No Binding 6.34.2 6 Binding Binding N.D. No Binding 6.67.15 Binding Binding N.D. Binding 6.73.2 3 Binding N.D. N.D. No Binding6.77.1 1 Binding Binding N.D. No Binding 9.8.2 3a Binding N.D. NoBinding No Binding

Anti-MAdCAM binding to specialised endothelial structures and lymphoidtissue is indicated by the shading, according to the key. The epitopebin based on Luminex epitope analysis and the pattern of MAdCAMcross-reactivity are indicated for each antibody. Luminex epitopebinning data for anti-MAdCAM antibodies 6.14.2, 6.22.2, 6.34.2, 6.67.1,6.73.3 and 6.77.1 (italics) were derived from separate experiments thanthat for 1.7.2, 1.8.2, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 (bold type), asindicated by the difference in font character.

All anti-MAdCAM antibodies tested had the ability to recognize a humanMAdCAM epitope expressed on vascular endothelial compartments of thegastrointestinal tract. Apart from 1.7.2 and 1.8.2, all otheranti-MAdCAM antibodies tested were able to specifically bind thevascular endothelial compartments of the cynomolgus gastrointestinaltract Certain other anti-MAdCAM antibodies, namely 6.14.2 and 6.67.1also had the ability to specifically recognize the dog MAdCAM orthologueas well as cynomolgus MAdCAM.

Generation of a Functionally Active Chimeric Cynomolgus/HumanMAdCAM-Expressing CHO Cell Line:

The differences in binding affinity of certain anti-MAdCAM antibodiesfor human and cynomolgus MAdCAM led us to determine whether a structuralbasis for this observation could be made.

Based on the published amino acid sequence for Macaque MAdCAM (Shyjan AM, et al., J Immunol., 156, 2851-7 (1996)), primers were designed to PCRamplify the cynomolgus MAdCAM α₄β₇ binding domain sequence. Total RNAwas prepared from frozen excised cynomolgus mesenteric lymph node (ca.200 mg) using the Trizol method (Invitrogen) according to themanufacturer's instructions. 1-2 μg was oligo-dT primed and reversetranscribed with AMV reverse transcriptase (Promega). A proportion ofthe reverse transcribed product was subjected to PCR with forward 5′-AGCATG GAT CGG GGC CTG GCC-3′ (SEQ ID NO: 67) and reverse 5′-GTG CAG GACCGG GAT GGC CTG-3′ (SEQ ID NO: 68) primers with GC-2 polymerase in 1M GCmelt (Clontech) and at an annealing temperature of 62° C. An RT-PCRproduct of the appropriate size was excised and purified from a 1%agarose gel after electrophoresis, then TOPO-TA cloned (Invitrogen)between EcoRI sites of pCR2.1. The insert was sequence confirmed. Thenucleotide and predicted translated amino acid sequences are shown inSEQ ID NOS 49 and 50, respectively.

The predicted human and cynomolgus MAdCAM amino acid sequences for theα₄β₇ ⁺ binding domain show a high degree of sequence identity (90.8%)when aligned (FIG. 3 provides this sequence alignment). To generate afunctionally active cynomolgus MAdCAM-expressing cell line, whichmimicked the anti-MAdCAM binding pattern represented by Table 8, a SacIfragment corresponding to the cynomolgus α₄β₇ ⁺ binding domain sequencein pCR2.1, was subcloned directly into the C-terminal human MAdCAMpIND-Hygro construct containing carboxyl-terminal mucin stalk andtransmembrane domain, described above. The sequence and orientation wasverified, then a KpnI/NotI fragment was cloned into pEF5FRTV5GWCATvector (Invitrogen), replacing the CAT coding sequence and used intransfections to generate single stably expressing clones in Flp In CHOcells (Invitrogen), according to the manufacturer's instructions.

The binding of anti-MAdCAM antibody clones to the CHO cells expressingcynomolgus/human MAdCAM chimera was assessed by flow cytometry and thefunctional activity of anti-MAdCAM antibodies was determined using avery similar JY cell adhesion assay as that described above. The bindingand functional activity of anti-MAdCAM antibodies are expressed in Table9.

TABLE 9 Correlation between the functional activity in thecynomolgus/human MAdCAM-CHO/JY adhesion assay and human andcynomolgus/human MAdCAM CHO cell binding, as measured by FACS, for arange of anti-MAdCAM antibodies. Functional IC₅₀ FACS binding Clone(μg/mL) human cyno/human IgG2 1.7.2 inactive Binding No Binding 1.8.2inactive Binding No Binding 7.16.6 0.72 Binding Binding 7.20.5 0.62Binding Binding 7.26.4 0.96 Binding Binding IgG4 6.14.2 0.53 BindingBinding 6.22.2 0.83 Binding Binding 6.34.2 0.47 Binding Binding 6.67.10.75 Binding Binding 6.73.2 Inactive Binding No Binding 6.77.1 0.64Binding Binding 9.8.2 0.83 Binding Binding

Taken together, there is a good correlation between the ability of agiven anti-MAdCAM antibody to bind human or cynomolgus MAdCAM, asdetected by immunohistochemistry (Table 8), with recombinant cell-basedbinding and functional activity (Table 9). Anti-MAdCAM antibodies 1.7.2,1.8.2 and 6.73.2, for instance, demonstrated a consistent lack ofbinding to cynomolgus tissue and cells expressing a chimericcynomolgus/human MAdCAM protein. Anti-MAdCAM antibodies 1.7.2, 1.8.2 and6.73.2 also did not have the ability to detect functional blockingactivity in the cynomolgus/human MAdCAM/JY adhesion assay.

Similar approaches could be used to define the epitope of theanti-MAdCAM antibodies 6.14.2 and 6.67.1 that recognise dog MAdCAM.

Example IV Use of Anti-MAdCAM mAbs in the Detection of CirculatingSoluble MAdCAM as a Method of Disease Diagnosis

Anti-MAdCAM antibodies can be used for the detection of circulatingsoluble MAdCAM (sMAdCAM). Detection of sMAdCAM in clinical plasma, serumsamples or other biofluid, such as, but not limited to, stool, urine,sputum. is likely to be a useful surrogate disease biomarker forunderlying disease, including, but not limited to, inflammatory boweldisease.

Based on the epitope binning data (Tables 7 and 8), anti-MAdCAMantibodies 1.7.2 and 7.16.6 appear to recognise different epitopes onhuman MAdCAM. ELISA plates were coated overnight at 4° C. with 100μL/well of a 50 μg/mL solution of 1.7.2 in phosphate buffered saline(PBS). After incubation the plate was blocked for 1.5 hours with a PBSblocking buffer containing 10% milk (200 μL/well). After incubation theplate was washed with PBS (2×100 μL/well) and serial dilutions ofMAdCAM-IgG1-Fc fusion protein, from a top concentration of 50 μg/mL downto approximately 5 ng/mL in PBS, to a final volume of 100 μL, were addedto the plate for incubation of 2 hours at room temperature. In a similarapproach the MAdCAM-IgG1-Fc protein can be diluted in plasma or serum,or some other such relevant biofluid and used to determine theexpression of soluble MAdCAM in a clinical sample, as described below.As a negative control, only buffer was added to the wells containing theprimary anti-MAdCAM antibody. After this time, the plate was washed withPBS (3×100 μL/well) and the plate then incubated in the dark with anAlexa488-labelled 7.16.6 (100 μL, 5 μg/mL). The Alexa488-labelled 7.16.6was generated using a commercially available kit (Molecular Probes,A-20181), following Manufacturer's protocols.

The plate was washed with PBS containing 0.05% Tween-20, and binding oflabeled 7.16.6 to captured soluble MAdCAM determined by measuring thefluorescence (Wallac Victor² 1420 Multilabel Reader, excitation X485 nm,emission X535 nm count from top, 3 mm from bottom of plate, for 0.1 secwith normal emission aperture). When fluorescence is plotted as afunction of the concentration of MAdCAM-IgG1-Fc fusion protein, FIG. 6,it indicates that 1.7.2 and a labeled 7.16.6 can be used for diagnosticpurposes to determine the level of circulating soluble MAdCAM expressedin a biofluid or clinical sample. This sandwich ELISA approach is notrestricted to the use of 1.7.2 and 7.16.6, but any combination ofanti-MAdCAM antibodies that recognise different epitopes on MAdCAM, asoutlined by the data and interpretation of table 7 and FIG. 5. Similarstrategies could be applied to the development of similar assays, suchas immunohistochemistry and Western Blot, with the other anti-MAdCAMantibodies described, using different partners, variants, labels, etc.

Example V Amino Acid Structure of Anti-MAdCAM mAbs Prepared inAccordance to the Invention

In the following discussion, structural information related to theanti-MAdCAM mAbs prepared in accordance with the invention is provided.

To analyze structures of mAbs produced in accordance with the invention,we cloned the genes encoding the heavy and light chain fragments out ofthe specific hybridoma clone. Gene cloning and sequencing wasaccomplished as follows:

Poly(A)+ mRNA was isolated from approximately 2×10⁵ hybridoma cellsderived from immunized XenoMouse mice using Fast-Track kit (Invitrogen).The generation of random primed cDNA was followed by PCR. Human VH or Vκfamily specific primers (Marks et al., ‘Oligonucleotide primers forpolymerase chain reaction amplification of human immunoglobulin variablegenese and design of family-specific oligonucleotide probes’; Eur. J.Immunol., 21, 985-991 (1991)) or a universal human VH primer, MG-30(5′-CAG GTG CAG CTG GAG CAG TCI GG-3 (SEQ ID NO: 108) was used inconjunction with primers specific for the human Cγ2, MG40-d (5′-GCT GAGGGA GTA GAG TCC TGA GGA-3 (SEQ ID NO: 109) or Cγ4 constant region,MG-40d (5′GCT GAG GGA GTA GAG TCC TGA GGA CTG T-3 (SEQ ID NO: 110), orCκ constant region (hκP2; as previously described in Green et al.,1994). Sequences of the human mAb-derived heavy and kappa chaintranscripts from hybridomas were obtained by direct sequencing of PCRproducts generated from poly (A+) RNA using the primers described above.PCR products were cloned into pCR2.1 using a TOPO-TA cloning kit(Invitrogen) and both strands were sequenced using Prism dye terminatorsequencing kits and an ABI 377 sequencing machine. All sequences wereanalysed by alignments to the ‘V BASE sequence directory’ (Tomlinson, etal, J. Mol. Biol., 227, 776-798 (1992); Hum. Mol. Genet., 3, 853-860(1994); EMBO J., 14, 4628-4638 (1995).)

Further each of the antibodies, 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2,6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod,6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod, were subjected tofull length DNA sequencing. For such, total RNA was isolated fromapproximately 3-6×10⁶ hybridoma cells using an RNeasy kit (Qiagen). ThemRNA was reverse transcribed using oligo-dT and an AMV-based reversetranscriptase system (Promega). V BASE was used to design 5′ specificamplification primers, containing an optimal Kozak sequence and ATGstart codon (underlined) and 3′ reverse primers for the specific heavyand kappa chains as depicted in Table 10.

TABLE 10 PCR primer pairs for cDNA amplification from anti-MAdCAM mAb-expressing hybridomas and primers used in the construction ofmodified versions of anti-MAdCAM antibodies. Oligo sequence VH1-18 5′TATCTAAGCTTCTAGACTCGAGCGCCACCATGGACTGGACCTGGAGCATCCTT 3′ (SEQ ID NO: 70)VH3-15 5′ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGATT 3′(SEQ ID NO: 71) VH3-21 5′TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAACTGGGGCTCCGCTGGGTT 3′ (SEQ ID NO: 72)VH3-23 5′ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGCTT 3′(SEQ ID NO: 73) VH3-30 5′TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGGTT 3′ (SEQ ID NO: 74)VH3-33 5′ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGGTT 3′(SEQ ID NO: 75) VH4-4 5′TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACACCTGTGGTTCTTCCTC 3′ (SEQ ID NO: 76)A2/A3 5′ TATCTAAGCTTCTAGACCCGGGCGCCACCATGAGGCTCCCTGCTCAGCTCCTG 3′(SEQ ID NO: 77) A26 5′TATCTAAGCTTCTAGACCCGGGCGCCACCATGTTGCCATCACAACTCATTGGG 3′ (SEQ ID NO: 78)B3 5′ TATCTAAGCTTCTAGACCCGGGCGCCACCATGGTGTTGCAGACCCAGGTCTTC 3′(SEQ ID NO: 79) O12 5′TATCTAAGCTTCTAGACCCGGGCGCCACCATGGACATGAGGGTCCCCGCTCAG 3′ (SEQ ID NO: 80)O18 5′ TATCTAAGCTTCTAGACCCGGGCGCCACCATGGACATGAGGGTCCCTGCTCAG 3′(SEQ ID NO: 81) RevIgG2 5′TTCTCTGATCAGAATTCCTATCATTTACCCGGAGACAGGGAGAG 3′ (SEQ ID NO: 82) RevIgG45′ TTCTTTGATCAGAATTCTCACTAACACTCTCCCCTGTTGAAGC 3′ (SEQ ID NO: 83)RevKappa 5′ TTCTCTGATCAGAATTCCTATCATTTACCCAGAGACAGGGAGAG 3′(SEQ ID NO: 84) 6.22.2VK_F15′-GGA TCT GGG ACA GAT TTC ACC CTC ACC ATC AAT AGC CTG GAA GC-3′(SEQ IDNO: 85) 6.22.2VK_R15′-GCT TCC AGG CTA TTG ATG GTG AGG GTG AAA TCT GTC CCA GAT CC-3′(SEQ IDNO: 86) 6.22.2VH_F15′-GCA GCG TCT GGA TTC ACC TTC AGT AGC-3′(SEQ ID NO: 87) 6.22.2VH_R15′-GCT ACT GAA GGT GAA TCC AGA CGC TGC-3′(SEQ ID NO: 88) 6.22.2VH_CS*5′-CGG AGG TGC TTC TAG AGC AGG GCG-3′(SEQ ID NO: 89) 6.34.2VK_F15′-GCA AGT CAG AGT ATT AGT AGC TAT TTA AAT TGG TAT CAG CAG AAA CC-3′(SEQ ID NO: 90) 6.34.2VK_R15′-GGT TTC TGC TGA TAC CAA TTT AAA TAG CTA CTA ATA CTC TGA CTT GC-3′(SEQ ID NO: 91) 6.34.2VK_F25′-CCA TCA GTT CTC TGC AAC CTG AGG ATT TTG CAA CTT ACT ACT GTC ACC-3′(SEQ ID NO: 92) 6.34.2VK_R35′-GGT GAC AGT AGT AAG TTG CAA AAT CCT CAG GTT GCA GAG AAC TGA TGG-3′(SEQ ID NO: 93) 6.34.2VH_F16.5′-GCA AAT GAA CAG CCT GCG CGC TGA GGA CAC G-3′(SEQ ID NO: 94) 34.2VH_R15′-CGT GTC CTC AGC GCG CAG GCT GTT CAT TTG C-3′(SEQ ID NO: 95)6.67.1VK_F15′-CAA TAA GAA CTA CTT AGC TTG GTA CCA ACA GAA ACC AGG ACA GCC-3′(SEQ ID NO: 96) 6.67.1VK_R15′-GGC TGT CCT GGT TTC TGT TGG TAC CAA GCT AAG TAG TTC TTA TTG-3′(SEQID NO: 97) 6.67.1VH_F15′-CCC TCA GGG GTC GAG TCA CCA TGT CAG TAG ACA CGT CCA AGA ACC-3′(SEQID NO: 98) 6.67.1VH_R15′-GGT TCT TGG ACG TGT CTA CTG ACA TGG TGA CTC GAC CCC TGA GGG-3′(SEQID NO: 99) 6.67.1VH_CS*5′-ATT CTA GAG CAG GGC GCC AGG-3′(SEQ ID NO: 100) 6.77.1VK_F15′-CCA TCT CCT GCA AGT CTA GTC AGA GCC TCC-3′(SEQ ID NO: 101)6.77.1VK_R15′-GGA GGC TCT GAC TAG ACT TGC AGG AGA TGG-3′(SEQ ID NO: 102)6.77.1VK_F25′-GGT TTA TTA CTG CAT GCA AAG TAT ACA GCT TAT GTC CAG TTT TGG CC -3′(SEQ ID NO: 103) 6.77.1VK_R25′-GGC CAA AAC TGG ACA TAA GCT GTA TAC TTT GCA TGC AGT AAT AAA CC -3′(SEQ ID NO: 104) 7.26.4K_F15′-CCT GCA AGT CTA GTC AGA GCC TCC-3′(SEQ ID NO: 105) 7.26.4K_R15′-GGA GGC TCT GAC TAG ACT TGC AGG-3′(SEQ ID NO: 106)

The primers pairs were used to amplify the cDNAs using Expand HighFidelity Taq polymerase (Roche), and the PCR products cloned into pCR2.1TOPO-TA (Invitrogen) for subsequent sequencing. Heavy and kappa lightchain sequence verified clones were then cloned into pEE6.1 and pEE12.1vectors (LONZA) using XbaI/EcoRI and HindIII/EcoRI sites respectively.

Gene Utilization Analysis

Table 11 displays the heavy and kappa light chain gene utilization foreach hybridoma outlined in the invention.

TABLE 11 Heavy and Kappa light chain Gene Utilization Kappa light HeavyChain Chain Clone VH D JH V_(K) J_(K) IgG2 1.7.2 VH 3-15 D6-19 JH4b A3JK5 1.8.2 VH 3-15 D6-19 JH4b A3 JK5 7.16.6 VH 1-18 D6-6 JH6b A2 JK17.20.5 VH4-4 D3-10 JH6b A3 JK4 7.26.4 VH1-18 D6-6 JH6b A2 JK1 IgG46.14.2 VH3-23 D5-5 JH4b O12 JK5 6.22.2 VH3-33 D5-12 JH6b A26 JK4 6.34.2VH3-30 D4-23 JH6b O12 JK3 6.67.1 VH4-4 D3-10 JH4b B3 JK4 6.73.2 VH3-23D6-19 JH6b O12 JK2 6.77.1 VH3-21 D6-19 JH6b A2 JK2 9.8.2 VH3-33 D3-10 orJH6b O18 JK5 D3-16

Sequence Analysis

To further examine antibody structure predicted amino acid sequences ofthe antibodies were obtained from the cDNAs obtained from the clones.

Sequence identifier numbers (SEQ ID NO:) 1-48 and 51-68 provide thenucleotide and amino acid sequences of the heavy and kappa light chainsof the anti-MAdCAM antibodies 1.7.2 (SEQ ID NOS 1-4), 1.8.2 (SEQ ID NOS5-8), 6.14.2 (SEQ ID NOS 9-12), 6.22.2 (SEQ ID NOS 13-16), 6.34.2 (SEQID NOS 17-20), 6.67.1 (SEQ ID NOS 21-24), 6.73.2 (SEQ ID NOS 25-28),6.77.1 (SEQ ID NOS 29-32), 7.16.6 (SEQ ID NOS 33-36), 7.20.5 (SEQ ID NOS37-40), 7.26.4 (SEQ ID NOS 41-44), 9.8.2 (SEQ ID NOS 45-48) and themodified anti-MAdCAM antibodies 6.22.2-mod (SEQ ID NOS 51-54),6.34.2-mod (SEQ ID NOS 55-58), 6.67.1-mod (SEQ ID NOS 59-62) and6.77.1-mod (SEQ ID NOS 63-66) and 7.26.4-mod (SEQ ID NOS 41-42, 67-68).For each anti-MAdCAM antibody sequence cloned, the sequences of thesignal peptide sequence (or the bases encoding the same) are indicatedin lower case and underlined.

FIGS. 1A-1J provide sequence alignments between the predicted heavychain amino acid sequences of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 and theamino acid sequence of the respective germline gene products. Thepositions of the CDR1, CDR2 and CDR3 sequences of the antibodies areunderlined, differences between the expressed sequence the correspondinggermline sequence are indicated in bold and where there are additions inthe expressed sequence compared to the germline these are indicated as a(−) in the germline sequence.

FIGS. 1K-1T provide sequence alignments between the predicted kappalight chain amino acid sequences of the antibodies 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2and the amino acid sequence of the respective germline gene products.The positions of the CDR1, CDR2 and CDR3 sequences of the antibodies areunderlined, differences between the expressed sequence the correspondinggermline they are indicated in bold and where there are additions in theexpressed sequence compared to the germline these are indicated as a (−)in the germline sequence.

Presence of Post-Translational Modification: Glycosylation andDeamidation:

The effect of some of the changes in the expressed anti-MAdCAM antibodysequence, compared with the derived germline sequence, is to introduceresidues that potentially could be subject to N-linked glycosylation(Asn-X-Ser/Thr) and/or deamidation (Asn-Gly) (see Table 12). The nucleicacid sequences encoding the kappa light chain variable domain amino acidsequences of the anti-MAdCAM antibodies 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.26.4 and 9.8.2, (SEQ ID NOS: 16, 20, 24, 28, 32, 44 and 48)and the heavy chain variable domain of antibody 6.14.2, (SEQ ID NO: 10),predict the presence of N-linked glycosylation. The presence of thispost-translational modification was investigated using a combination ofSDS-PAGE and Pro-Q® Emerald 488 Glycoprotein (Molecular Probes) stainingwith mAbs 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.26.4 and 9.8.2.

Briefly, approximately 2 μg of reduced anti-MAdCAM antibody was loadedonto a 4-12% SDS-polyacrylamide gel using a MOPS buffer. Followingelectrophoresis, the gel was fixed in 50% MeOH, 5% acetic acid andwashed in 3% acetic acid. Any carbohydrates on the gel were thenoxidised with periodic acid and stained using Pro-Q® Emerald 488Glycoprotein Stain Kit (Molecular Probes). After a final wash step,glycoprotein staining was visualised using a fluorescence scanner set ata wavelength of 473 nm.

After glycoprotein staining, the gel was stained for total protein usingSYPRO Ruby protein gel stain and analysed using a fluorescence scannerset at a wavelength of 473 nm. The kappa light chains of anti-MAdCAMantibodies, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.26.4 and 9.8.2,all stained positively for the presence of glycosylation. As anadditional confirmation, anti-MAdCAM antibody 7.26.4, was subjected totryptic/chymotrypic digestion, the LC-MS/MS analysis confirmed thepresence of a modified tryptic peptide and provided additionalconfirmation of kappa light chain glycosylation.

Specific Asn-Gly sequences in the CDR1 regions of anti-MAdCAMantibodies, 1.7.2, 1.8.2, 6.22.2 and 7.20.5, render these regionssensitive to deamidation. Deamidation at neutral pH introduces anegative charge and can also lead to β-isomerisation, which could affectthe properties of an antibody. For anti-MAdCAM antibodies 1.7.2, 1.8.2and 7.20.5, the presence of deamidated Asn-isoaspartate residues wasassessed by mass spectroscopy following trapping the isoaspartate sidechain with MeOH.

In brief, for the anti-MAdCAM antibody 1.7.2, the status of thetryptic/Asp-N peptide SSQSLLQSNGYNYL (SEQ ID NO: 69) (1573.7 Da) wasselected for monitoring by LC-MS/MS. Anti-MAdCAM antibody 1.7.2 wasreduced in 10 mM DTT, alkylated in 5 mM Na iodoacetate and subsequentlybuffer exchanged into trypsin digestion buffer (50 mM Tris-HCl, 1 mMCaCl₂, pH 7.6). The antibody was then mixed with sequencing grademodified trypsin (Promega) in a protease:protein ratio of 1:20. Proteinwas digested in trypsin for 15 hours at 30° C., and the resultingpeptides separated by HPLC using a C-18 RPC on an Ettan LC system. The³³Asn-containing peptide (4032 Da) was collected from the column anddiluted in Asp-N digestion buffer (50 mM sodium phosphate buffer, pH8.0). Endoproteinase Asp-N(Roche) was then added at an approximatepeptide:enzyme ratio of 10:1.

Acetyl chloride (100 μL) was added to a sample of methanol (1 mL, −20°C.), the mixture warmed to room temperature. The tryptic+Asp-N digestwas dried in a Speed-Vac and then 5 μL of the methanol/acetyl chloridewas added (45 mM, room temp), then dried again in a Speed-Vac. Theresulting residue was re-constituted in 0.1% TFA and peptides wereanalysed initially on the Voyager-DE STR MALDI-TOF mass spectrometerusing either the nitrocellulose thin layer sample preparation method orreverse phase purification using C18 ZipTips (Millipore) followed bydroplet mixing with α-cyano matrix. The methylated peptide mixture wasalso analysed using LC-MS/MS on a Deca XP Plus Ion Trap MassSpectrometer as above. The elution was plumbed straight into the IonTrap MS and peptides were subsequently analysed by MS and MS/MS. The MSwas set to analyse all ions between 300 and 2000 Da. The strongest ionin any particular scan was then subjected to MS/MS analysis.

TABLE 12 Post-translational modification of anti-MAdCAM antibodiesHeavy Chain Kappa light chain Glycosylation Glyrosylation DeamidationClone (NXS/T) Confirmed (NXS/T) Confirmed (NG) Confirmed IgG2 1.7.2LQSNGYN MS 1.8.2 LQSNGYN MS 7.16.6 7.20.5 HGNGYNY MS 7.26.4 CKSNQSLLYMS / PAGE IgG4 6.14,2 TFNNSAMT N.D. | 6.22.2 SGTNFTLTI PAGE LTINGLEA N.D6.34.2 ASQNISSYL PAGE 6.67.1 SSNNKTYLA PAGE 6.73.2 RASQNITN PAGE 6.77.1SCNSSQSL PAGE 9.8.2 HSDNLSIT PAGE

Mutagenesis Studies:

The primary amino acid sequence of the anti-MAdCAM antibodiesexemplified in this invention can be modified, by site-directedmutagenesis, to remove potential sites of post-translationalmodification (e.g., glycosylation, de-amidation) or to alter the isotypebackground, or to engineer other changes which may improve thetherapeutic utility. As an example, PCR was used to engineer changes tothe anti-MAdCAM antibodies 6.22.2, 6.34.2, 6.67.1, 6.77.1 and 7.26.4, torevert certain framework sequences to germline, to remove potentialglycosylation sites and/or to change the isotype background to a humanIgG₂. pCR2.1 TOPO-TA cloned cDNAs (100 ng), corresponding to heavy chainnucleotide SEQ ID NOS: 13, 17, 21 and 29, and kappa light nucleotide SEQID NOS: 15, 19, 23, 31 and 43, were used as a template in a series ofPCRs using overlap-extension and a panel of primer sets described inTable 10.

6.22.2 Heavy Chain:

PCR primer sets 6.22.2_VH_F1 and 6.22.2VH_CS* (1) and VH3-33 and6.22.2_VH_R1 (2) were used to generate separate PCR products (1) and(2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template(100 ng) represented by nucleotide sequence SEQ ID NO: 13. Products (1)and (2) were purified and combined in a third PCR step (ca. 50 ng each)along with VH3-33 and VK6.22.2_CS* primers, to generate the modified6.22.2 heavy chain V-domain. This modified version contains a His/Phemutation in FR1 and introduces an XbaI restriction site to enable inframe cloning into a pEE6.1 derived vector, termed pEE6.1CH, whichcontains the corresponding human IgG₂ constant domain. The final PCRfragment was cloned into the XbaI site of pEE6.1CH, checked fororientation and the insert full sequence verified. The nucleotidesequence for the modified 6.22.2 heavy chain is found in SEQ ID NO: 51and the corresponding amino acid sequence in SEQ ID NO: 52. The changesin the nucleotide and amino acid sequences compared with the parent areindicated.

6.22.2 Kappa Light Chain:

PCR primer sets 6.22.2_VK_F1 and revKappa (1), and A26 and 6.22.2_VK_R1(2) were used to generate separate PCR products (1) and (2), using anExpand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng)represented by nucleotide sequence SEQ ID NO: 15. Products (1) and (2)were purified and combined in a third PCR step (ca. 50 ng each) alongwith A26 and revKappa primers, to generate the modified 6.22.2 kappalight chain V-domain. This modified version contains Asn/Asp and Gly/Serchanges to the FR3 sequence. The resultant PCR product was cloned intopEE12.1 using HindIII/EcoR1 sites and fully sequence verified. Thenucleotide sequence for the modified 6.22.2 kappa light chain is foundin SEQ ID NO: 53 and the corresponding amino acid sequence in SEQ ID NO:54. The changes in the nucleotide and amino acid sequences compared withthe parent are indicated.

6.34.2 Heavy Chain:

PCR primer sets 6.34.2_VH_F1 and 6.22.2VH_CS* (1) and VH3-30 and6.34.2_VH_R1 (2) were used to generate separate PCR products (1) and(2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template(100 ng) represented by nucleotide sequence SEQ ID NO: 17. Products (1)and (2) were purified and combined in a third PCR step (ca. 50 ng each)along with VH3-30 and VK6.22.2_CS* primers, to generate the modified6.34.2 heavy chain V-domain. This modified version contains a Ser/Argmutation in FR3 and introduces an XbaI restriction site to enable inframe cloning into a pEE6.1 derived vector, termed pEE6.1CH, whichcontains the corresponding human IgG₂ constant domain. The final PCRfragment was cloned into the XbaI site of pEE6.1CH, checked fororientation and the insert full sequence verified. The nucleotidesequence for the modified 6.34.2 heavy chain is found in SEQ ID NO: 55and the corresponding amino acid sequence in SEQ ID NO: 56. The changesin the nucleotide and amino acid sequences compared with the parent areindicated.

6.34.2 Kappa Light Chain:

PCR primer sets 012 and 6.34.2_VK_R1 (1), 6.34.2_VK_F1 and 6.34.2_VK_R2(2), as well as 6.34.2_VK_F2 and revKappa (3) were used to generateseparate PCR products (1), (2) and (3), using an Expand Taq polymeraseand a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucleotidesequence SEQ ID NO: 19. Products (1), (2) and (3) were purified and (1)and (2) were combined in a third PCR step (ca. 50 ng each), along with012 and 6.34.2_VK_R2 primers, to generate the PCR product (4). PCRproducts (2) and (3) were combined in a fourth PCR step (ca. 50 ngeach), along with 6.34.2_VK_F1 and revKappa, to generate the PCR product(5). PCR products (4) and (5) were purified and combined together (ca.50 ng each) with primers 012 and revKappa to generate the modified6.34.2 kappa light chain V-domain. This modified version contains anAsn/Ser change in CDR1, a Phe/Tyr change in FR2 and Arg-Thr/Ser-Ser,Asp/Glu and Ser/Tyr changes to the FR3 sequence. The resultant PCRproduct was cloned into pEE12.1 using HindIII/EcoR1 sites and fullysequence verified. The nucleotide sequence for the modified 6.34.2 kappalight chain is found in SEQ ID NO: 57 and the corresponding amino acidsequence in SEQ ID NO: 58. The changes in the nucleotide and amino acidsequences compared with the parent are indicated.

6.67.1 Heavy Chain:

PCR primer sets 6.67.1_VH_F1 and 6.67.1VH_CS* (1) and VH4-4 and6.67.1_VH_R1 (2) were used to generate separate PCR products (1) and(2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template(100 ng) represented by nucleotide sequence SEQ ID NO: 21. Products (1)and (2) were purified and combined in a third PCR step (ca. 50 ng each)along with VH4-4 and VK6.67.1_CS* primers, to generate the modified6.67.1 heavy chain V-domain. This modified version contains anIle-Leu-Ala/Met-Ser-Val conversion in FR3 and introduces an XbaIrestriction site to enable in frame cloning into a pEE6.1 derivedvector, termed pEE6.1CH, which contains the corresponding human IgG₂constant domain. The final PCR fragment was cloned into the XbaI site ofpEE6.1CH, checked for orientation and the insert full sequence verified.The nucleotide sequence for the modified 6.67.1 heavy chain is found inSEQ ID NO: 59 and the corresponding amino acid sequence in SEQ ID NO:60. The changes in the nucleotide and amino acid sequences compared withthe parent are indicated.

6.67.1 Kappa Light Chain:

PCR primer sets 6.67.1_VK_F1 and revKappa (1), and B3 and 6.67.1_VK_R1(2) were used to generate separate PCR products (1) and (2), using anExpand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng)represented by nucleotide sequence SEQ ID NO: 23. Products (1) and (2)were purified and combined in a third PCR step (ca. 50 ng each) alongwith B3 and revKappa primers, to generate the modified 6.67.1 kappalight chain V-domain. This modified version contains a Thr/Asn change inCDR1 and an Arg/Gly change in FR2. The resultant PCR product was clonedinto pEE12.1 using HindIII/EcoR1 sites and fully sequence verified. Thenucleotide sequence for the modified 6.67.1 kappa light chain is foundin SEQ ID NO: 61 and the corresponding amino acid sequence in SEQ ID NO:62. The changes in the nucleotide and amino acid sequences compared withthe parent are indicated.

6.77.1 Heavy Chain:

PCR primer sets VH 3-21 and 6.22.2VH_CS* were used to generate a singlePCR product using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNAtemplate (100 ng) represented by nucleotide sequence SEQ ID NO: 29. ThePCR products were digested with XbaI, gel purified and cloned into theXbaI site of pEE6.1CH, checking for orientation. The insert was fullysequence verified. The nucleotide sequence for the modified 6.77.1 heavychain is found in SEQ ID NO: 63 and the corresponding amino acidsequence in SEQ ID NO: 64. The changes in the nucleotide and amino acidsequences compared with the parent are indicated. 6.77.1 Kappa LightChain:

PCR primer sets A2 and 6.77.1_VK_R1 (1), 6.77.1_VK_VK_F1 and 6.77.1_R2(2), as well as 6.77.1_VK_F2 and revKappa (3) were used to generateseparate PCR products (1), (2) and (3), using an Expand Taq polymeraseand a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucleotidesequence SEQ ID NO: 31. Products (1), (2) and (3) were purified and, (1)and (2) were combined in a third PCR step (ca. 50 ng each) along with A2and 6.77.1_VK_R2 primers, to generate PCR product (4). PCR product (2)and (3) were combined in a fourth PCR step (ca. 50 ng each) along with6.77.1_VK_F1 and revKappa primers, to generate PCR product (5). PCRproducts (4) and (5) were purified and combined together (ca. 50 ngeach) with primers A2 and JK2 to generate the modified 6.77.1 kappalight chain V-domain. This modified version contains an Asn/Lys changein CDR1, a Ser/Tyr change in FR3 and a Cys/Ser residue change in CDR3sequence. The resultant PCR product was cloned into pEE12.1 usingHindIII/EcoR1 sites and fully sequence verified. The nucleotide sequencefor the modified 6.77.1 kappa light chain is found in SEQ ID NO: 65 andthe corresponding amino acid sequence in SEQ ID NO: 66. The changes inthe nucleotide and amino acid sequences compared with the parent areindicated.

7.26.4 Kappa Light Chain:

PCR primer sets 7.26.4_VK_F1 and revKappa (1), and A2 and 7.26.4_VK_R1(2) were used to generate separate PCR products (1) and (2), using anExpand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng)represented by nucleotide sequence SEQ ID NO: 43. Products (1) and (2)were purified and combined in a third PCR step (ca. 50 ng each) alongwith A2 and revKappa primers, to generate the modified 7.26.4 kappalight chain V-domain. This modified version contains an Asn/Ser changein CDR1. The resultant PCR product was cloned into pEE12.1 usingHindIII/EcoR1 sites and fully sequence verified. The nucleotide sequencefor the modified 7.26.4 kappa light chain is found in SEQ ID NO: 67 andthe corresponding amino acid sequence in SEQ ID NO: 68. The changes inthe nucleotide and amino acid sequences compared with the parent areindicated.

A functional eukaryotic expression vector for each of the modifiedversions of 6.22.2, 6.34.2, 6.67.1, 6.77.1 and 7.26.4, referred to as6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod, andrepresenting respectively the heavy chain nucleotide sequences SEQ IDNOS: 51, 55, 59, 63 and 41, and corresponding amino acid sequences SEQID NOS: 52, 56, 60, 64 and 42, as well as the kappa light chainnucleotide sequences SEQ ID NOS: 53, 57, 61, 65 and 67, and thecorresponding amino acid sequences SEQ ID NOS: 54, 58, 62, 66 and 68were assembled as follows: The heavy chain cDNA inserts corresponding to6.22.2-mod, 6.34.2-mod, 6.67.1-mod and 6.77.1-mod were excised from thepEE6.1CH vector with NotI/SalI, the parental version of the heavy chainsof 7.26.4 was excised from the pEE6.1 vector with NotI/SalI, and thepurified fragments were cloned into identical sites into thecorresponding pEE12.1 vector containing the modified versions of thekappa light chain sequences 6.22.2-mod, 6.34.2-mod, 6.67.1-mod,6.77.1-mod and 7.26.4-mod. The sequences of the vectors were confirmed,and purified amounts used in transient transfections with HEK 293Tcells. Briefly, 9×10⁶ HEK 293T cells, seeded in a T165 flask the daybefore transfection and washed into Optimem, were transientlytransfected with vector cDNAs corresponding to 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod and 7.26.4-mod (40 μg) using Lipofectamine PLUS(Invitrogen) according to manufacturer's instructions. The cells wereincubated for 3 hrs, then the transfection media replaced with DMEM(Invitrogen 21969-035) media containing 10% ultra-low IgG fetal calfserum (Invitrogen 16250-078) and L-Glutamine (50 mL). The mediasupernatant was harvested 5 days later, filter sterilised and theanti-MAdCAM antibody purified using protein G sepharose affinitychromatography, in a similar manner as to that described above. Theamount of antibody recovered (20-100 μg) was quantified by a Bradfordassay.

The anti-MAdCAM activity of affinity purified antibody corresponding to6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod wasassessed in the MAdCAM-IgG1-Fc fusion assay as described previously. TheIC₅₀ values of these anti-MADCAM antibodies compared with the parentalanti-MAdCAM antibodies from which they were derived are presented inTable 13. There was minimal effect of the amino acid substitutionsdescribed above on the activity of the modified anti-MAdCAM antibodiescompared with their parents was minimal. The antibodies also maintainedtheir binding to CHO cells expressing recombinant human MAdCAM or thecynomolgus/human MAdCAM chimera.

TABLE 13 Activity of modified versions of anti-MAdCAM antibodies,6.22.2- mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod comparedwith their parents. MAdCAM IgG1 Fc fusion Assay Mean IC50 (μg/mL) CLONEParent Modified 6.22.2 0.018 0.058 6.34.2 0.013 0.049 6.67.1 0.013 0.0376.77.1 0.022 0.077 7.26.4 0.021 0.033

Example VI Increase in β₇ ⁺ Lymphocytes in the Peripheral Circulation byBlocking Anti-MAdCAM Antibodies

An assay was developed to identify and correlate a mechanistic effect ofan anti-MAdCAM antibody and its circulating level in blood. Aninhibitory anti-MAdCAM antibody should have the effect of inhibiting therecruitment of leukocytes expressing the α₄β₇ ⁺ integrin to thegastrointestinal tract. Classes of α₄β₇ integrin-bearing leukocytesshould, therefore, be restricted to the peripheral circulation

This was demonstrated with a fully human anti-human MAdCAM mAb 7.16.6,in cynomolgus.

Purified anti-human MAdCAM mAb 7.16.6 (1 mg/kg) or vehicle (20 mMNaAcetate, 0.2 mg/mL polysorbate 80, 45 mg/mL mannitol, and 0.02 mg/mLEDTA at pH 5.5) were assessed in a similar manner by intravenousadministration via the saphenous vein to two groups of cynomolgusmonkeys (n=4/group). At day 3 post-dosing blood samples were collectedin EDTA tubes by femoral venipuncture. LPAM specific antibodies, whichcrossreact with the cynomolgus α₄β₇ ⁺ integrin, are not commerciallyavailable, so an anti-β₇ antibody (recognising α₄β₇ and α_(E)β₇integrin) was used instead. Antibodies (30 μL), according to thefollowing table, table 15, were added to tubes containing 100 μL ofcynomolgus blood, mixed by gentle vortexing and incubated for 20-30 minsat 4° C.

TABLE 15 Antibodies (BD Pharmingen) used in immunophenotyping ofcynomologus blood Catalogue Number Antibody or Isotype 555748 mIgG1,k-FITC 555844 mIgG2a, k-PE 559425 mIgG1-PerCP 555751 mIgG1, k-APC 555728CD 28-FITC 555945 β7-PE 558814 CD 95-APC 550631 CD 4-PerCP

To each tube, 1 mL of 1:10 FACSlyse solution (BD #349202) was added,mixed by gentle vortex and incubated at room temperature forapproximately 12 minutes in the dark until red blood cell lysis wascomplete. Then 2 mL of BD stain buffer (#554656) was added to each tube,mixed and centrifuged at 250×g for 6-7 mins at room temperature. Thesupernatant was decanted and the pellet resuspended in 3 mL of stainbuffer, mixed again and centrifuged at 250×g for 6-7 mins at roomtemperature. Cytofix buffer (BD #554655), containing w/vparaformaldehyde (100 μL) was added to the cell pellets from monkeyperipheral blood and mixed thoroughly by low/moderate speed of vortexer.The samples were kept at 4° C. in the dark until they acquired on theFACSCalibur. Just prior to acquisition, PBS (100 μL) was added to alltubes immediately before acquisition. The absolute cell numbers ofCD4⁺β₇ ⁺CD95loCD28⁺ (naïve), CD4⁺β₇ ⁺CD95hiCD28⁺ (central memory),CD4⁺β₇-CD95hiCD28⁺ (central memory), CD4⁺β₇ ⁺CD95hiCD28⁻ (effectormemory) were acquired by appropriate gating and quandrant analyses.Other T cell subsets for example, CD8⁺ T central memory cell (β₇⁺CD8⁺CD28⁺CD95⁺) and any other leukocytes bearing a MAdCAM ligand, mayalso be analyzed by this method with the appropriate antibodies.Compared with the vehicle control, anti-MAdCAM mAb 7.16.6 caused anapproximate 3 fold increase in the levels of circulating CD4⁺β₇⁺CD95hiCD28⁺ central memory T cells, as shown in FIG. 7. There were noeffects on the population of circulating CD4⁺β₇-CD95hiCD28⁺ centralmemory T cells, indicating that the effect of anti-MAdCAM mAb 7.16.6 isspecific for gut homing T cells. The effects of anti-MAdCAM mAb 7.16.6,in cynomolgus, on populations of circulating (α₄)β₇ ⁺ lymphocytesindicates that this is a robust surrogate proof of mechanism biomarker,particularly in the context of practical application in a clinicalsetting.

Sequences

SEQ ID NO: 1-48 and 51-68 provide nucleotide and amino acid sequences ofthe heavy and kappa light chains for twelve human anti-MAdCAMantibodies, nucleotide and amino acid sequences of cynomolgus MAdCAMα₄β₇ binding domain sequences and nucleotide and amino acid sequences offive modified human anti-MAdCAM antibodies.

SEQ ID NO: 1-48 provide the heavy and kappa light chain nucleotide andamino acid sequences of twelve human monoclonal anti-MAdCAM antibodies:1.7.2 (SEQ ID NO: 1-4), 1.8.2 (SEQ ID NO: 5-8), 6.14.2 (SEQ ID NO:9-12), 6.22.2 (SEQ ID NO: 13-16), 6.34.2 (SEQ ID NO: 17-20), 6.67.1 (SEQID NO: 21-24), 6.73.2 (SEQ ID NO: 25-28), 6.77.1 (SEQ ID NO: 29-32),7.16.6 (SEQ ID NO: 33-36), 7.20.5 (SEQ ID NO: 37-40), 7.26.4 (SEQ ID NO:41-44), and 9.8.2 (SEQ ID NO: 45-48).

SEQ ID NO: 49-50 provide the nucleotide and amino acid sequences of acynomolgus MAdCAM α₄β₇ binding domain.

SEQ ID NO: 51-68 provide the heavy and kappa light chain nucleotide andamino acid sequences for the modified monoclonal anti-MAdCAM antibodies:6.22.2 (SEQ ID NO: 51-54), modified 6.34.2 (SEQ ID NO: 55-58), modified6.67.1 (SEQ ID NO: 59-62), modified 6.77.1 (SEQ ID NO: 63-66) and thekappa light chain nucleotide and amino acid sequences of modifiedmonoclonal anti-MAdCAM antibody: modified 7.26.4 (SEQ ID NO: 67-68).

SEQ ID NOS: 70-106 and 108-110 provide various primer sequences.

Key:

Signal sequence: underlined lower caseAmino acid changes in modified anti-MAdCAM antibodies sequence comparedto parent: underlined upper case

SEQ ID NO. 1 1.7.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg gattttcctt gctgctattt taaaaggtgt 51ccagtgtGAG GTGCAGCTGG TGGAGTCTGG GGGAGGCTTG GTGAAGCCTG 101GGGGGTCCCT TAGACTCTCC TGTGTAGCCT CTGGATTCAC TTTCACTAAC 151GCCTGGATGA TCTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT 201TGGCCGTATT AAAAGGAAAA CTGATGGTGG GACAACAGAC TACGCTGCAC 251CCGTGAAAGG CAGATTCACC ATCTCAAGAG ATGATTCAAA AAACACGCTG 301TATCTGCAAA TGAACAGCCT GAAAACCGAG GACACAGCCG TGTATTACTG 351TACCACAGGG GGAGTGGCTG AGGACTACTG GGGCCAGGGA ACCCTGGTCA 401CCGTCTCCTC AGCCTCCACC AAGGGCCCAT CGGTCTTCCC CCTGGCGCCC 451TGCTCCAGGA GCACCTCCGA GAGCACAGCG GCCCTGGGCT GCCTGGTCAA 501GGACTACTTC CCCGAACCGG TGACGGTGTC GTGGAACTCA GGCGCTCTGA 551CCAGCGGCGT GCACACCTTC CCAGCTGTCC TACAGTCCTC AGGACTCTAC 601TCCCTCAGCA GCGTGGTGAC CGTGCCCTCC AGCAACTTCG GCACCCAGAC 651CTACACCTGC AACGTAGATC ACAAGCCCAG CAACACCAAG GTGGACAAGA 701CAGTTGAGCG CAAATGTTGT GTCGAGTGCC CACCGTGCCC AGCACCACCT 751GTGGCAGGAC CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT 801CATGATCTCC CGGACCCCTG AGGTCACGTG CGTGGTGGTG GACGTGAGCC 851ACGAAGACCC CGAGGTCCAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG 901CATAATGCCA AGACAAAGCC ACGGGAGGAG CAGTTCAACA GCACGTTCCG 951TGTGGTCAGC GTCCTCACCG TTGTGCACCA GGACTGGCTG AACGGCAAGG 1001AGTACAAGTG CAAGGTCTCC AACAAAGGCC TCCCAGCCCC CATCGAGAAA 1051ACCATCTCCA AAACCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT 1101GCCCCCATCC CGGGAGGAGA TGACCAAGAA CCAGGTCAGC CTGACCTGCC 1151TGGTCAAAGG CTTCTACCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT 1201GGGCAGCCGG AGAACAACTA CAAGACCACA CCTCCCATGC TGGACTCCGA 1251CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC 1301AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC 1351CACTACACGC AGAAGAGCCT CTCCCTGTCT CCGGGTAAAT GA SEQ ID NO. 21.7.2 Predicted Heavy Chain Protein Sequence 1mefglswifl aailkgvqcE VQLVESGGGL VKPGGSLRLS CVASGFTFTN 51AWMIWVRQAP GKGLEWVGRI KRKTDGGTTD YAAPVKGRFT ISRDDSKNTL 101YLQMNSLKTE DTAVYYCTTG GVAEDYWGQG TLVTVSSAST KGPSVFPLAP 151CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY 201SLSSVVTVPS SNFGTQTYTC NVDHKPSNTK VDKTVERKCC VECPPCPAPP 251VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ FNWYVDGVEV 301HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS NKGLPAPIEK 351TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN 401GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN 451HYTQKSLSLS PGK SEQ ID NO. 3 1.7.2 Kappa Light Chain Nucleotide Sequence1 atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtctctgg 51atccagtggg GATATTGTGA TGACTCAGTC TCCACTCTCC CTGCCCGTCA 101CCCCTGGAGA GCCGGCCTCC ATCTCCTGCA GGTCTAGTCA GAGCCTCCTG 151CAAAGTAATG GATACAACTA TTTGGATTGG TACCTGCAGA AGCCAGGGCA 201GTCTCCACAG CTCCTGATCT ATTTGGGTTC TAATCGGGCC TCCGGGGTCC 251CTGACAGGTT CAGTGGCAGT GGATCAGGCA CAGATTTTAC ACTGAAAATC 301AGCAGAGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAGCTCT 351ACAAACTATC ACCTTCGGCC AAGGGACACG ACTGGAGATT AAACGAACTG 401TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA 451TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA 501GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC 551AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC 601AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC 651CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA 701ACAGGGGAGA GTGTTAGTGA SEQ ID NO. 41.7.2 Predicted Kappa Light Chain Protein Sequence 1mrlpaqllgl lmlwvsgssg DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL 51QSNGYNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI 101SRVEAEDVGV YYCMQALQTI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK 151SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 202STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC SEQ ID NO. 51.8.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg gattttcctt gctgctattt taaaaggtgt 51ccagtgtGAG GTGCAGCTGG TGGAGTCTGG GGGAGGCTTG GTGAAGCCTG 101GGGGGTCCCT TAGACTCTCC TGTGTAGTCT CTGGATTCAC TTTCACTAAC 151GCCTGGATGA TCTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT 201TGGCCGTATT AAAAGGAAAA CTGATGGTGG GACAACAGAC TACGCTGCAC 251CCGTGAAAGG CAGATTCACC ATCTCAAGAG ATGATTCAAA AAACACGCTG 301TATCTGCAAA TGAACAGCCT GAAAACCGAG GACACAGCCG TGTATTACTG 351TACCACAGGG GGAGTGGCTG AGGACTACTG GGGCCAGGGA ACCCTGGTCA 401CCGTCTCCTC AGCCTCCACC AAGGGCCCAT CGGTCTTCCC CCTGGCGCCC 451TGCTCCAGGA GCACCTCCGA GAGCACAGCG GCCCTGGGCT GCCTGGTCAA 501GGACTACTTC CCCGAACCGG TGACGGTGTC GTGGAACTCA GGCGCTCTGA 551CCAGCGGCGT GCACACCTTC CCAGCTGTCC TACAGTCCTC AGGACTCTAC 601TCCCTCAGCA GCGTGGTGAC CGTGCCCTCC AGCAACTTCG GCACCCAGAC 651CTACACCTGC AACGTAGATC ACAAGCCCAG CAACACCAAG GTGGACAAGA 701CAGTTGAGCG CAAATGTTGT GTCGAGTGCC CACCGTGCCC AGCACCACCT 751GTGGCAGGAC CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT 801CATGATCTCC CGGACCCCTG AGGTCACGTG CGTGGTGGTG GACGTGAGCC 851ACGAAGACCC CGAGGTCCAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG 901CATAATGCCA AGACAAAGCC ACGGGAGGAG CAGTTCAACA GCACGTTCCG 951TGTGGTCAGC GTCCTCACCG TTGTGCACCA GGACTGGCTG AACGGCAAGG 1001AGTACAAGTG CAAGGTCTCC AACAAAGGCC TCCCAGCCCC CATCGAGAAA 1051ACCATCTCCA AAACCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT 1101GCCCCCATCC CGGGAGGAGA TGACCAAGAA CCAGGTCAGC CTGACCTGCC 1151TGGTCAAAGG CTTCTACCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT 1201GGGCAGCCGG AGAACAACTA CAAGACCACA CCTCCCATGC TGGACTCCGA 1251CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC 1301AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC 1351CACTACACGC AGAAGAGCCT CTCCCTGTCT CCGGGTAAAT GA SEQ ID NO. 61.8.2 Predicted Heavy Chain Protein Sequence 1mefglswifl aailkgvqcE VQLVESGGGL VKPGGSLRLS CVVSGFTFTN 51AWMIWVRQAP GKGLEWVGRI KRKTDGGTTD YAAPVKGRFT ISRDDSKNTL 101YLQMNSLKTE DTAVYYCTTG GVAEDYWGQG TLVTVSSAST KGPSVFPLAP 151CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY 201SLSSVVTVPS SNFGTQTYTC NVDHKPSNTK VDKTVERKCC VECPPCPAPP 251VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ FNWYVDGVEV 301HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS NKGLPAPIEK 351TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN 401GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN 451HYTQKSLSLS PGK SEQ ID NO. 7 1.8.2 Kappa Light Chain Nucleotide Sequence1 atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtctctgg 51atccagtggg GATATTGTGA TGACTCAGTC TCCACTCTCC CTGCCCGTCA 101CCCCTGGAGA GCCGGCCTCC ATCTCCTGCA GGTCTAGTCA GAGCCTCCTG 151CAAAGTAATG GATTCAACTA TTTGGATTGG TACCTGCAGA AGCCAGGGCA 201GTCTCCACAG CTCCTGATCT ATTTGGGTTC TAATCGGGCC TCCGGGGTCC 251CTGACAGGTT CAGTGGCAGT GGGTCAGGCA CAGATTTTAC ACTGAAAATC 301AGCAGAGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAGCTCT 351ACAAACTATC ACCTTCGGCC AAGGGACACG ACTGGAGATT AAACGAACTG 401TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA 451TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA 501GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC 551AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC 601AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC 651CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA 701ACAGGGGAGA GTGTTAGTGA SEQ ID NO. 81.8.2 Predicted Kappa Light Chain Protein Sequence 1mrlpaqllgl lmlwvsgssg DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL 51QSNGFNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI 101SRVEAEDVGV YYCMQALQTI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK 151SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 202STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC SEQ ID NO. 96.14.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt 51ccagtgtGAG GTGCAGCTGT TGGAGTCTGG GGGAGGCTTG GTACAGCCTG 101GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGACTCAC CTTTAACAAT 151TCTGCCATGA CCTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT 201CTCAACTACT AGTGGAAGTG GTGGTACCAC ATACTACGCA GACTCCGTGA 251AGGGCCGGTT CACCATCTCC AGAGACTCTC CCAAGAACAC GCTCTATCTG 301CAAATGAACA GCCTGAGAGC CGAGGACACG GCCGTATATT ACTGTGCGGC 351CCGTGGATAC AGCTATGGTA CGACCCCCTA TGAGTACTGG GGCCAGGGAA 401CCCTGGTCAC CGTCTCCTCA GCTTCCACCA AGGGCCCATC CGTCTTCCCC 451CTGGCGCCCT GTTCCAGGAG CACCTCCGAG AGCACAGCCG CCCTGGGCTG 501CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG 551GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA 601GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG 651CACGAAGACC TACACCTGCA ACGTAGATCA CAAGCCCAGC AACACCAAGG 701TGGACAAGAG AGTTGAGTCC AAATATGGTC CCCCATGCCC ATCATGCCCA 751GCACCTGAGT TCCTGGGGGG ACCATCAGTC TTCCTGTTCC CCCCAAAACC 801CAAGGACACT CTCATGATCT CCCGGACCCC TGAGGTCACG TGCGTGGTGG 851TGGACGTGAG CCAGGAAGAC CCCGAGGTCC AGTTCAACTG GTACGTGGAT 901GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTTCAA 951CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC 1001TGAACGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGG CCTCCCGTCC 1051TCCATCGAGA AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAGCCACA 1101GGTGTACACC CTGCCCCCAT CCCAGGAGGA GATGACCAAG AACCAGGTCA 1151GCCTGACCTG CCTGGTCAAA GGCTTCTACC CCAGCGACAT CGCCGTGGAG 1201TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT 1251GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAGGCTA ACCGTGGACA 1301AGAGCAGGTG GCAGGAGGGG AATGTCTTCT CATGCTCCGT GATGCATGAG 1351GCTCTGCACA ACCACTACAC ACAGAAGAGC CTCTCCCTGT CTCTGGGTAA 1401 ATGASEQ ID NO. 10 6.14.2 Predicted Heavy Chain Protein Sequence 1mefglswlfl vailkgvqcE VQLLESGGGL VQPGGSLRLS CAASGLTFNN 51SAMTWVRQAP GKGLEWVSTT SGSGGTTYYA DSVKGRFTIS RDSPKNTLYL 101QMNSLRAEDT AVYYCAARGY SYGTTPYEYW GQGTLVTVSS ASTKGPSVFP 151LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 201GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP 251APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 301GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 351SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 401WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 451ALHNHYTQKS LSLSLGK SEQ ID NO. 116.14.2 Kappa Light Chain Nucleotide Sequence 1atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct 51ccgaggggcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT 101CTGCATCTGT AGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCGGAGC 151ATTAGCAGCT ATTTAAATTG GTATCAGCAG AAACCAGGGA AAGCCCCTAA 201AGTCCTGATC TTTTTTGTGT CCAGTTTGCA AAGTGGGGTC CCATCAAGGT 251TCAGTGGCAG TGGCTCTGGG ACAGATTTCA CTCTCACCAT CAGCAGTCTG 301CAACCTGAAG ATTTTGCAAC TTACTACTGT CAACAGAATT ACATTCCCCC 351TATTACCTTC GGCCAGGGGA CACGACTGGA GATCAGACGA ACTGTGGCTG 401CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA 451ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA 501AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA 551GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC 601CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA 651AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG 701 GAGAGTGTTA GSEQ ID NO. 12 6.14.2 Predicted Kappa Light Chain Protein Sequence 1mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASRS 51ISSYLNWYQQ KPGKAPKVLI FFVSSLQSGV PSRFSGSGSG TDFTLTISSL 101QPEDFATYYC QQNYIPPITF GQGTRLEIRR TVAAPSVFIF PPSDEQLKSG 151TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 202LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO. 136.22.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt 51ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG 101GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGACACAC CTTCAGTAGC 151GATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT 201GGCAATTATA TGGTATGATG GAAGTAATAA ATATTATGCA GACTCCGTGA 251AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG 301CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTATATT ACTGTGCGAG 351AGATCCCGGC TACTATTACG GTATGGACGT CTGGGGCCAA GGGACCACGG 401TCACCGTCTC CTCAGCTTCC ACCAAGGGCC CATCCGTCTT CCCCCTGGCG 451CCCTGCTCCA GGAGCACCTC CGAGAGCACA GCCGCCCTGG GCTGCCTGGT 501CAAGGACTAC TTCCCCGAAC CGGTGACGGT GTCGTGGAAC TCAGGCGCCC 551TGACCAGCGG CGTGCACACC TTCCCGGCTG TCCTACAGTC CTCAGGACTC 601TACTCCCTCA GCAGCGTGGT GACCGTGCCC TCCAGCAGCT TGGGCACGAA 651GACCTACACC TGCAACGTAG ATCACAAGCC CAGCAACACC AAGGTGGACA 701AGAGAGTTGA GTCCAAATAT GGTCCCCCAT GCCCATCATG CCCAGCACCT 751GAGTTCCTGG GGGGACCATC AGTCTTCCTG TTCCCCCCAA AACCCAAGGA 801CACTCTCATG ATCTCCCGGA CCCCTGAGGT CACGTGCGTG GTGGTGGACG 851TGAGCCAGGA AGACCCCGAG GTCCAGTTCA ACTGGTACGT GGATGGCGTG 901GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT TCAACAGCAC 951GTACCGTGTG GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAACG 1001GCAAGGAGTA CAAGTGCAAG GTCTCCAACA AAGGCCTCCC GTCCTCCATC 1051GAGAAAACCA TCTCCAAAGC CAAAGGGCAG CCCCGAGAGC CACAGGTGTA 1101CACCCTGCCC CCATCCCAGG AGGAGATGAC CAAGAACCAG GTCAGCCTGA 1151CCTGCCTGGT CAAAGGCTTC TACCCCAGCG ACATCGCCGT GGAGTGGGAG 1201AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCGCGCCTC CCGTGCTGGA 1251CTCCGACGGC TCCTTCTTCC TCTACAGCAG GCTAACCGTG GACAAGAGCA 1301GGTGGCAGGA GGGGAATGTC TTCTCATGCT CCGTGATGCA TGAGGCTCTG 1351CACAACCACT ACACACAGAA GAGCCTCTCC CTGTCTCTGG GTAAATGA SEQ ID NO. 146.22.2 Predicted Heavy Chain Protein Sequence 1mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGHTFSS 51DGMHWVRQAP GKGLEWVAII WYDGSNKYYA DSVKGRFTIS RDNSKNTLYL 101QMNSLRAEDT AVYYCARDPG YYYGMDVWGQ GTTVTVSSAS TKGPSVFPLA 151PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 201YSLSSVVTVP SSSLGTKTYT CNVDHKPSNT KVDKRVESKY GPPCPSCPAP 251EFLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV 301EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI 351EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE 401SNGQPENNYK TAPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL 451HNHYTQKSLS LSLGK SEQ ID NO. 156.22.2 Kappa Light Chain Nucleotide Sequence 1atgttgccat cacaactcat tgggtttctg ctgctctggg ttccagcttc 51caggggtGAA ATTGTGCTGA CTCAGTCTCC AGACTTTCAG TCTGTGACTC 101CAAAAGAGAA AGTCACCATC ACCTGCCGGG CCAGTCAGAG AATTGGTAGT 151AGCTTACACT GGTACCAGCA GAAACCAGAT CAGTCTCCAA AACTCCTCAT 201CAAGTATGCT TCCCAGTCCT TCTCAGGGGT CCCCTCGAGG TTCAGTGGCA 251GTGGATCTGG GACAAATTTC ACCCTCACCA TCAATGGCCT GGAAGCTGAA 301GATGCTGCAA CTTATTACTG TCATCAGAGT GGTCGTTTAC CGCTCACTTT 351CGGCGGAGGG ACCAAGGTGG AGATCAAACG AACTGTGGCT GCACCATCTG 401TCTTCATCTT CCCGCCATCT GATGAGCAGT TGAAATCTGG AACTGCCTCT 451GTTGTGTGCC TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG 501GAAGGTGGAT AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG 551AGCAGGACAG CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG 601AGCAAAGCAG ACTACGAGAA ACACAAAGTC TACGCCTGCG AAGTCACCCA 651TCAGGGCCTG AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGTT 701 AGTGASEQ ID NO. 16 6.22.2 Predicted Kappa Light Chain Protein Sequence 1mlpsqligfl llwvpasrgE IVLTQSPDFQ SVTPKEKVTI TCRASQRIGS 51SLHWYQQKPD QSPKLLIKYA SQSFSGVPSR FSGSGSGTNF TLTINGLEAE 101DAATYYCHQS GRLPLTFGGG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS 151VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 201SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC SEQ ID NO. 176.34.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt 51ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG 101GGAGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC 151TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT 201GGCAGTTATA TCAAATGATG GAAATAATAA ATACTATGCA GACTCCGTGA 251AGGGCCGATT CACCATCTCC AGAGACAATT CCAAAAACAC GCTGTATCTG 301CAAATGAACA GCCTGAGCGC TGAGGACACG GCTGTGTATT ACTGTGCGAG 351AGATAGTACG GCGATAACCT ACTACTACTA CGGAATGGAC GTCTGGGGCC 401AAGGGACCAC GGTCACCGTC TCCTCAGCTT CCACCAAGGG CCCATCCGTC 451TTCCCCCTGG CGCCCTGCTC CAGGAGCACC TCCGAGAGCA CAGCCGCCCT 501GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA 551ACTCAGGCGC CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAG 601TCCTCAGGAC TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAG 651CTTGGGCACG AAGACCTACA CCTGCAACGT AGATCACAAG CCCAGCAACA 701CCAAGGTGGA CAAGAGAGTT GAGTCCAAAT ATGGTCCCCC ATGCCCATCA 751TGCCCAGCAC CTGAGTTCCT GGGGGGACCA TCAGTCTTCC TGTTCCCCCC 801AAAACCCAAG GACACTCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG 851TGGTGGTGGA CGTGAGCCAG GAAGACCCCG AGGTCCAGTT CAACTGGTAC 901GTGGATGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA 951GTTCAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG 1001ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC 1051CCGTCCTCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA 1101GCCACAGGTG TACACCCTGC CCCCATCCCA GGAGGAGATG ACCAAGAACC 1151AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201GTGGAGTGGG AGAGCAATGG ACAGCCGGAG AACAACTACA AGACCACGCC 1251TCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AGGCTAACCG 1301TGGACAAGAG CAGGTGGCAG GAGGGGAATG TCTTCTCATG CTCCGTGATG 1351CATGAGGCTC TGCACAACCA CTACACACAG AAGAGCCTCT CCCTGTCTCT 1401 GGGTAAATGASEQ ID NO. 18 6.34.2 Predicted Heavy Chain Protein Sequence 1mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS 51YGMHWVRQAP GKGLEWVAVI SNDGNNKYYA DSVKGRFTIS RDNSKNTLYL 101QMNSLSAEDT AVYYCARDST AITYYYYGMD VWGQGTTVTV SSASTKGPSV 151FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ 201SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPS 251CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY 301VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL 351PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA 401VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM 451HEALHNHYTQ KSLSLSLGK SEQ ID NO. 196.34.2 Kappa Light Chain Nucleotide Sequence 1atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct 51ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT 101CTGCATCTGT CGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCAGAAT 151ATTAGTAGCT ATTTAAATTG GTTTCAGCAG AAACCAGGGA AAGCCCCTAA 201GCTCCTGATC TATGCTGCAT CCGGTTTGAA GCGTGGGGTC CCATCACGGT 251TCAGTGGTAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGGACTCTG 301CAACCTGATG ATTTTGCAAC TTACTCCTGT CACCAGAGTT ACAGTCTCCC 351ATTCACTTTC GGCCCTGGGA CCAAAGTGGA TATCAAACGA ACTGTGGCTG 401CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA 451ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA 501AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA 551GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC 601CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA 651AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG 701GAGAGTGTTA GTGA SEQ ID NO. 206.34.2 Predicted Kappa Light Chain Protein Sequence 1mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASQN 51ISSYLNWFQQ KPGKAPKLLI YAASGLKRGV PSRFSGSGSG TDFTLTIRTL 101QPDDFATYSC HQSYSLPFTF GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG 151TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 201LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO. 216.67.1 Heavy Chain Nucleotide Sequence 1atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt 51cctgtccCAG GTGCAGCTGC AGGAGTCGGG CCCAGGACTG GTGAAGCCTT 101CGGAGACCCT GTCCCTCACC TGCACTGTCT CTGGTGACTC CATCAGTAGT 151AACTATTGGA GCTGGATCCG GCAGCCCGCC GGGAAGGGAC TGGAGTGGAT 201TGGGCGTATC TATACCAGTG GGGGCACCAA CTCCAACCCC TCCCTCAGGG 251GTCGAGTCAC CATTTTAGCA GACACGTCCA AGAACCAGTT CTCTCTGAAA 301CTGAGTTCTG TGACCGCCGC GGACACGGCC GTGTATTACT GTGCGAGAGA 351TCGTATTACT ATAATTCGGG GACTTATTCC ATCCTTCTTT GACTACTGGG 401GCCAGGGAAC CCTGGTCACC GTCTCCTCAG CTTCCACCAA GGGCCCATCC 451GTCTTCCCCC TGGCGCCCTG CTCCAGGAGC ACCTCCGAGA GCACAGCCGC 501CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT 551GGAACTCAGG CGCCCTGACC AGCGGCGTGC ACACCTTCCC GGCTGTCCTA 601CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG 651CAGCTTGGGC ACGAAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA 701ACACCAAGGT GGACAAGAGA GTTGAGTCCA AATATGGTCC CCCATGCCCA 751TCATGCCCAG CACCTGAGTT CCTGGGGGGA CCATCAGTCT TCCTGTTCCC 801CCCAAAACCC AAGGACACTC TCATGATCTC CCGGACCCCT GAGGTCACGT 851GCGTGGTGGT GGACGTGAGC CAGGAAGACC CCGAGGTCCA GTTCAACTGG 901TACGTGGATG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA 951GCAGTTCAAC AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC 1001AGGACTGGCT GAACGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGGC 1051CTCCCGTCCT CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG 1101AGAGCCACAG GTGTACACCC TGCCCCCATC CCAGGAGGAG ATGACCAAGA 1151ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTACCC CAGCGACATC 1201GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC 1251GCCTCCCGTG CTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAGGCTAA 1301CCGTGGACAA GAGCAGGTGG CAGGAGGGGA ATGTCTTCTC ATGCTCCGTG 1351ATGCATGAGG CTCTGCACAA CCACTACACA CAGAAGAGCC TCTCCCTGTC 1401TCTGGGTAAA TGA SEQ ID NO. 226.67.1 Predicted Heavy Chain Protein Sequence 1mkhlwfflll vaaprwvlsQ VQLQESGPGL VKPSETLSLT CTVSGDSISS 51NYWSWIRQPA GKGLEWIGRI YTSGGTNSNP SLRGRVTILA DTSKNQFSLK 101LSSVTAADTA VYYCARDRIT IIRGLIPSFF DYWGQGTLVT VSSASTKGPS 151VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL 201QSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP 251SCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW 301YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG 351LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI 401AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV 451MHEALHNHYT QKSLSLSLGK SEQ ID NO. 236.67.1 Kappa Light Chain Nucleotide Sequence 1atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg 51tgcctacggg GACATCGTGA TGACCCAGTC TCCAGACTCC CTGGCTGTGT 101CTCTGGGCGA GAGGGCCACC ATCAACTGCA AGTCCAGCCA GAGTGTTTTA 151TACAGCTCCA ACAATAAGAC CTACTTAGCT TGGTACCAAC AGAAACCAAG 201ACAGCCTCCT AAATTGCTCA TTTACTGGGC ATCTATACGG GAATATGGGG 251TCCCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC 301ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTTTATTTCT GTCAACAATA 351TTATAGTATT CCTCCCCTCA CTTTCGGCGG AGGGACCAAG GTGGAGATCA 401AACGAACTGT GGCTGCACCA TCTGTCTTCA TCTTCCCGCC ATCTGATGAG 451CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA ATAACTTCTA 501TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG 551GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC 601AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA 651AGTCTACGCC TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA 701AGAGCTTCAA CAGGGGAGAG TGTTAGTGA SEQ ID NO. 246.67.1 Predicted Kappa Light Chain Protein Sequence 1mylqtqvfis lllwisgayg DIVMTQSPDS LAVSLGERAT INCKSSQSVL 51YSSNNKTYLA WYQQKPRQPP KLLIYWASIR EYGVPDRFSG SGSGTDFTLT 101ISSLQAEDVA VYFCQQYYSI PPLTFGGGTK VEIKRTVAAP SVFIFPPSDE 151QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY 201SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C SEQ ID NO. 256.73.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt 51ccagtgtGAG GTGCAGCTGT TGGAGTCTGG GGGAGACTTG GTCCAGCCTG 101GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTTAGAAGT 151TATGCCATGA ACTGGGTCCG ACAGGCTCCA GGGAAGGGGC TGGAGTGGGT 201CTCAGTTATT AGTGGTCGTG GTGGTACTAC ATACTACGCA GACTCCGTGA 251AGGGCCGGTT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG 301CAAATGAACA GCCTGAGAGC CGAGGACGCG GCCGTATATT ACTGTGCGAA 351GATAGCAGTG GCTGGAGAGG GGCTCTACTA CTACTACGGT ATGGACGTCT 401GGGGCCAAGG GACCACGGTC ACCGTCTCCT CAGCTTCCAC CAAGGGCCCA 451TCCGTCTTCC CCCTGGCGCC CTGCTCCAGG AGCACCTCCG AGAACACAGC 501CGCCCTGGGC TGCCTGGTCA AGGACTACTT CCCCGAACCG GTGACGGTGT 551CGTGGAACTC AGGCGCCCTG ACCAGCGGCG TGCACACCTT CCCGGCTGTC 601CTACAGTCCT CAGGACTCTA CTCCCTCAGC AGCGTGGTGA CCGTGCCCTC 651TAGCAGCTTG GGCACGAAGA CCTACACCTG CAACGTAGAT CACAAGCCCA 701GCAACACCAA GGTGGACAAG AGAGTTGAGT CCAAATATGG TCCCCCATGC 751CCATCATGCC CAGCACCTGA GTTCCTGGGG GGACCATCAG TCTTCCTGTT 801CCCCCCAAAA CCCAAGGACA CTCTCATGAT CTCCCGGACC CCTGAGGTCA 851CGTGCGTGGT GGTGGACGTG AGCCAGGAAG ACCCCGAGGT CCAGTTCAAC 901TGGTACGTGG ATGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA 951GGAGCAGTTC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC 1001ACCAGGACTG GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 1051GGCCTCCCGT CCTCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC 1101CCGAGAGCCA CAGGTGTACA CCCTGCCCCC ATCCCAGGAG GAGATGACCA 1151AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC 1201ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC 1251CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAGGC 1301TAACCGTGGA CAAGAGCAGG TGGCAGGAGG GGAATGTCTT CTCATGCTCC 1351GTGATGCATG AGGCTCTGCA CAACCACTAC ACACAGAAGA GCCTCTCCCT 1401GTCTCTGGGT AAATGATAG SEQ ID NO. 266.73.2 Predicted Heavy Chain Protein Sequence 1mefglswlfl vailkgvqcE VQLLESGGDL VQPGGSLRLS CAASGFTFRS 51YAMNWVRQAP GKGLEWVSVI SGRGGTTYYA DSVKGRFTIS RDNSKNTLYL 101QMNSLRAEDA AVYYCAKIAV AGEGLYYYYG MDVWGQGTTV TVSSASTKGP 151SVFPLAPCSR STSENTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV 201LQSSGLYSLS SVVTVPSSSL GTKTYTCNVD HKPSNTKVDK RVESKYGPPC 251PSCPAPEFLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SQEDPEVQFN 301WYVDGVEVHN AKTKPREEQF NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK 351GLPSSIEKTI SKAKGQPREP QVYTLPPSQE EMTKNQVSLT CLVKGFYPSD 401IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSRLTVDKSR WQEGNVFSCS 451VMHEALHNHY TQKSLSLSLG K SEQ ID NO. 276.73.2 Kappa Light Chain Nucleotide Sequence 1atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct 51ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT 101CTGCATCTGT AGGTGACAGA GTCACCTTCA CTTGCCGGGC AAGTCAGAAC 151ATTACCAACT ATTTAAATTG GTATCAGCAG AAACCAGGGA AGGCCCCTAA 201GCTCCTGATC TATGCTGCGT CCAGTTTGCC AAGAGGGGTC CCATCAAGGT 251TCCGTGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGTCTG 301CAACCTGAAG ATTTTGCAAC TTACTACTGT CAACAGAGTT ACAGTAATCC 351TCCGGAGTGC GGTTTTGGCC AGGGGACCAC GCTGGATATC AAACGAACTG 401TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA 451TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA 501GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC 551AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC 601AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC 651CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA 701ACAGGGGAGA GTGTTAGTGA SEQ ID NO. 286.73.2 Predicted Kappa Light Chain Protein Sequence 1mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTFTCRASQN 51ITNYLNWYQQ KPGKAPKLLI YAASSLPRGV PSRFRGSGSG TDFTLTISSL 101QPEDFATYYC QQSYSNPPEC GFGQGTTLDI KRTVAAPSVF IFPPSDEQLK 151SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 201STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC SEQ ID NO. 296.77.1 Heavy Chain Nucleotide Sequence 1atggaactgg ggctccgctg ggttttcctt gttgctattt tagaaggtgt 51ccagtgtGAG GTGCAGCTGG TGGAGTCTGG GGGAGGCCTG GTCAAGCCTG 101GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC 151TATAGCATGA ACTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT 201CTCATCCATT AGTAGTAGTA GTAGTTACAT ATACTACGCA GACTCAGTGA 251AGGGCCGATT CACCATCTCC AGAGACAACG CCAAGAACTC ACTGTATCTG 301CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG 351AGATGGGTAT AGCAGTGGCT GGTCCTACTA CTACTACTAC GGTATGGACG 401TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAGCTTC CACCAAGGGC 451CCATCCGTCT TCCCCCTGGC GCCCTGCTCC AGGAGCACCT CCGAGAGCAC 501AGCCGCCCTG GGCTGCCTGG TCAAGGACTA CTTCCCCGAA CCGGTGACGG 551TGTCGTGGAA CTCAGGCGCC CTGACCAGCG GCGTGCACAC CTTCCCGGCT 601GTCCTACAGT CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACCGTGCC 651CTCCAGCAGC TTGGGCACGA AGACCTACAC CTGCAACGTA GATCACAAGC 701CCAGCAACAC CAAGGTGGAC AAGAGAGTTG AGTCCAAATA TGGTCCCCCA 751TGCCCATCAT GCCCAGCACC TGAGTTCCTG GGGGGACCAT CAGTCTTCCT 801GTTCCCCCCA AAACCCAAGG ACACTCTCAT GATCTCCCGG ACCCCTGAGG 851TCACGTGCGT GGTGGTGGAC GTGAGCCAGG AAGACCCCGA GGTCCAGTTC 901AACTGGTACG TGGATGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG 951GGAGGAGCAG TTCAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC 1001TGCACCAGGA CTGGCTGAAC GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 1051AAAGGCCTCC CGTCCTCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA 1101GCCCCGAGAG CCACAGGTGT ACACCCTGCC CCCATCCCAG GAGGAGATGA 1151CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTACCCCAGC 1201GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA 1251GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA 1301GGCTAACCGT GGACAAGAGC AGGTGGCAGG AGGGGAATGT CTTTTCACGC 1351TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACACAGA AGAGCCTCTC 1401CCTGTCTCTG GGTAAATGAT AGGAATTCTG ATGA SEQ ID NO. 306.77.1 Predicted Heavy Chain Protein Sequence 1melglrwvfl vailegvqcE VQLVESGGGL VKPGGSLRLS CAASGFTFSS 51YSMNWVRQAP GKGLEWVSSI SSSSSYIYYA DSVKGRFTIS RDNAKNSLYL 101QMNSLRAEDT AVYYCARDGY SSGWSYYYYY GMDVWGQGTT VTVSSASTKG 151PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA 201VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP 251CPSCPAPEFL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF 301NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN 351KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS 401DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSR 451SVMHEALHNH YTQKSLSLSL GK SEQ ID NO. 316.77.1 Kappa Light Chain Nucleotide Sequence 1atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg 51atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA 101CTCCTGGACA GCCGGCCTCC ATCTCCTGCA ACTCTAGTCA GAGCCTCCTG 151CTTAGTGATG GAAAGACCTA TTTGAATTGG TACCTGCAGA AGCCCGGCCA 201GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC 251CAGACAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC 301AGCCGGGTGG AGGCTGAGGA TGTTGGGGTT TATTCCTGCA TGCAAAGTAT 351ACAGCTTATG TGCAGTTTTG GCCAGGGGAC CAAGCTGGAG ATCAAACGAA 401CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG 451AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG 501AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT 551CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC 601AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA 651CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT 701TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO. 326.77.1 Predicted Kappa Light Chain Protein Sequence 1mrlpagllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCNSSQSLL 51LSDGKTYLNW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI 101SRVEAEDVGV YSCMQSIQLM CSFGQGTKLE IKRTVAAPSV FIFPPSDEQL 151KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 201SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO. 337.16.6 Heavy Chain Nucleotide Sequence 1atggactgga cctggagcat ccttttcttg gtggcagcag caacaggtgc 51ccactccCAG GTTCAGCTGG TGCAGTCTGG AGCTGAGGTG AAGAAGCCTG 101GGGCCTCAGT GAAGGTCTCC TGCAAGGCTT CTGGTTACAC CTTTACCAGC 151TATGGTATCA ACTGGGTGCG ACAGGCCCCT GGACAAGGGC TTGAGTGGAT 201GGGATGGATC AGCGTTTACA GTGGTAACAC AAACTATGCA CAGAAGGTCC 251AGGGCAGAGT CACCATGACC GCAGACACAT CCACGAGCAC AGCCTACATG 301GACCTGAGGA GCCTGAGATC TGACGACACG GCCGTGTATT ACTGTGCGAG 351AGAGGGTAGC AGCTCGTCCG GAGACTACTA TTACGGTATG GACGTCTGGG 401GCCAAGGGAC CACGGTCACC GTCTCCTCAG CCTCCACCAA GGGCCCATCG 451GTCTTCCCCC TGGCGCCCTG CTCCAGGAGC ACCTCCGAGA GCACAGCGGC 501CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT 551GGAACTCAGG CGCTCTGACC AGCGGCGTGC ACACCTTCCC AGCTGTCCTA 601CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG 651CAACTTCGGC ACCCAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA 701ACACCAAGGT GGACAAGACA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA 751CCGTGCCCAG CACCACCTGT GGCAGGACCG TCAGTCTTCC TCTTCCCCCC 801AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG 851TGGTGGTGGA CGTGAGCCAC GAAGACCCCG AGGTCCAGTT CAACTGGTAC 901GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCAC GGGAGGAGCA 951GTTCAACAGC ACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGG 1001ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC 1051CCAGCCCCCA TCGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA 1101ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC 1151AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC 1251TCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 1301TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 1351CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 1401 GGGTAAATGASEQ ID NO. 34 7.16.6 Predicted Heavy Chain Protein Sequence 1mdwtwsilfl vaaatgahsQ VQLVQSGAEV KKPGASVKVS CKASGYTFTS 51YGINWVRQAP GQGLEWMGWI SVYSGNTNYA QKVQGRVTMT ADTSTSTAYM 101DLRSLRSDDT AVYYCAREGS SSSGDYYYGM DVWGQGTTVT VSSASTKGPS 151VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL 201QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP 251PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY 301VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL 351PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA 401VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM 451HEALHNHYTQ KSLSLSPGK SEQ ID NO. 357.16.6 Kappa Light Chain Nucleotide Sequence 1atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg 51atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA 101CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG 151CATACTGATG GAACGACCTA TTTGTATTGG TACCTGCAGA AGCCAGGCCA 201GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC 251CAGATAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC 301AGCCGGGTGG AGGCTGAGGA TGTTGGGATT TATTACTGCA TGCAAAATAT 351ACAGCTTCCG TGGACGTTCG GCCAAGGGAC CAAGGTGGAA ATCAAACGAA 401CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG 451AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG 501AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT 551CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC 601AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA 651CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT 701TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO. 367.16.6 Kappa Light Chain Protein Sequence 1mrlpaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL 51HTDGTTYLYW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI 101SRVEAEDVGI YYCMQNIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL 151KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 201SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO. 377.20.5 Heavy Chain Nucleotide Sequence 1atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt 51cctgtccCAG GTGCAGCTGC AGGAGTCGGG CCCAGGACTG GTGAAGCCTT 101CGGAGACCCT GTCCCTCACC TGCACTGTCT CTGGTAGCTC CATCAGTAGT 151TACCACTGGA ACTGGATCCG GCAGCCCGCC GGGAAGGGAC TGGAGTGGAT 201TGGGCGTATC TATACCAGTG GGAGCACCAA CTACAACCCC TCCCTCAAGA 251GTCGAGTCAC CATGTCACTA GACACGTCCA AGAACCAGTT CTCCCTGAAG 301CTGAGCTCTG TGACCGCCGC GGACACGGCC GTGTATTACT GTGCGAGAGA 351GGGGGTCAGG TATTACTATG CTTCGGGGAG TTATTACTAC GGTCTGGACG 401TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAGCCTC CACCAAGGGC 451CCATCGGTCT TCCCCCTGGC GCCCTGCTCC AGGAGCACCT CCGAGAGCAC 501AGCGGCCCTG GGCTGCCTGG TCAAGGACTA CTTCCCCGAA CCGGTGACGG 551TGTCGTGGAA CTCAGGCGCT CTGACCAGCG GCGTGCACAC CTTCCCAGCT 601GTCCTACAGT CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACCGTGCC 651CTCCAGCAAC TTCGGCACCC AGACCTACAC CTGCAACGTA GATCACAAGC 701CCAGCAACAC CAAGGTGGAC AAGACAGTTG AGCGCAAATG TTGTGTCGAG 751TGCCCACCGT GCCCAGCACC ACCTGTGGCA GGACCGTCAG TCTTCCTCTT 801CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA 851CGTGCGTGGT GGTGGACGTG AGCCACGAAG ACCCCGAGGT CCAGTTCAAC 901TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCACGGGA 951GGAGCAGTTC AACAGCACGT TCCGTGTGGT CAGCGTCCTC ACCGTTGTGC 1001ACCAGGACTG GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 1051GGCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAACCA AAGGGCAGCC 1101CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA 1151AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC 1201ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC 1251CACACCTCCC ATGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC 1301TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC 1351GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT 1401GTCTCCGGGT AAATGA SEQ ID NO. 387.20.5 Predicted Heavy Chain Protein Sequence 1mkhlwfflll vaaprwvlsQ VQLQESGPGL VKPSETLSLT CTVSGSSISS 51YHWNWIRQPA GKGLEWIGRI YTSGSTNYNP SLKSRVTMSL DTSKNQFSLK 101LSSVTAADTA VYYCAREGVR YYYASGSYYY GLDVWGQGTT VTVSSASTKG 151PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA 201VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE 251CPPCPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN 301WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK 351GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD 401IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS 451VMHEALHNHY TQKSLSLSPG K SEQ ID NO. 397.20.5 Kappa Light Chain Nucleotide Sequence 1atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtctctgg 51atccagtggg GATATTGTGA TGACTCAGTC TCCACTCTCC CTGCCCGTCA 101CCCCTGGAGA GCCGGCCTCC ATCTCCTGCA GGTCTAGTCA GAGCCTCCTG 151CATGGTAATG GATACAACTA TTTGGATTGG TACCTGCAGA AGCCAGGGCA 201GTCTCCACAG CTCCTGATCT ATTTGGGTTC TAATCGGGCC TCCGGGGTCC 251CTGACAGGTT CAGTGGCAGT GGATCAGGCA CAGATTTTAC ACTGAAAATC 301AGCAGAGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAGCTCT 351ACAAACTCTC ACTTTCGGCG GAGGGACCAA GGTGGAGATC AAACGAACTG 401TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA 451TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA 501GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC 551AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC 601AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC 651CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA 701ACAGGGGAGA GTGTTAGTGA SEQ ID NO. 407.20.5 Predicted Kappa Light Chain Protein Sequence 1mrlpaqllgl lmlwvsgssg DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL 51HGNGYNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI 101SRVEAEDVGV YYCMQALQTL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK 151SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 201STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC SEQ ID NO. 417.26.4 Heavy Chain Nucleotide Sequence 1atggactgga cctggagcat ccttttcttg gtggcagcag caacaggtgc 51ccactccCAG GTTCAGCTGG TGCAGTCTGG AGCTGAGGTG AAGAAGCCTG 101GGGCCTCAGT GAAGGTCTCC TGCGAGGCTT CTGGTTACAC CTTTACCAGC 151TATGGTATCG ACTGGGTGCG ACAGGCCCCT GGACAAGGGC TTGAGTGGAT 201GGGATGGATC AGCGTTTACA GTGGTAACAC AAACTATGCA CAGAAGCTCC 251AGGGCAGAGT CACCATGTCC ACAGACACAT CCACGAGCAC AGCCTACATG 301GAGCTGAGGA GCCTGAGATC TGACGACACG GCCGTGTATT ACTGTGCGAG 351AGAGGGTAGC AGCTCGTCCG GAGACTACTA CTACGGTATG GACGTCTGGG 401GCCAAGGGAC CACGGTCACC GTCTCCTCAG CCTCCACCAA GGGCCCATCG 451GTCTTCCCCC TGGCGCCCTG CTCCAGGAGC ACCTCCGAGA GCACAGCGGC 501CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT 551GGAACTCAGG CGCTCTGACC AGCGGCGTGC ACACCTTCCC AGCTGTCCTA 601CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG 651CAACTTCGGC ACCCAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA 701ACACCAAGGT GGACAAGACA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA 751CCGTGCCCAG CACCACCTGT GGCAGGACCG TCAGTCTTCC TCTTCCCCCC 801AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG 851TGGTGGTGGA CGTGAGCCAC GAAGACCCCG AGGTCCAGTT CAACTGGTAC 901GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCAC GGGAGGAGCA 951GTTCAACAGC ACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGG 1001ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC 1051CCAGCCCCCA TTGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA 1101ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC 1151AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC 1251TCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 1301TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 1351CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 1402 GGGTAAATGASEQ ID NO. 42 7.26.4 Predicted Heavy Chain Protein Sequence 1mdwtwsilfl vaaatgahsQ VQLVQSGAEV KKPGASVKVS CEASGYTFTS 51YGIDWVRQAP GQGLEWMGWI SVYSGNTNYA QKLQGRVTMS TDTSTSTAYM 101ELRSLRSDDT AVYYCAREGS SSSGDYYYGM DVWGQGTTVT VSSASTKGPS 151VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL 201QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP 251PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY 301VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL 351PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA 401VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM 451HEALHNHYTQ KSLSLSPGK SEQ ID NO. 437.26.4 Kappa Light Chain Nucleotide Sequence 1atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg 51atccagtgcg GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA 101CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAATCA GAGCCTCCTG 151TATAGTGATG GAAAGACCTA TTTGTTTTGG TACCTGCAGA AGCCAGGCCA 201GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGATTC TCTGGAGTGC 251CAGATAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC 301AGCCGGGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAAGTAT 351ACAGCTTCCG TGGACGTTCG GCCAAGGGAC CAAGGTGGAA ATCAAACGAA 401CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG 451AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG 501AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT 551CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC 601AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA 651CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT 701TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO. 447.26.4 Predicted Kappa Light Chain Protein Sequence 1mrlpaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSNQSLL 51YSDGKTYLFW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI 101SRVEAEDVGV YYCMQSIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL 151KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 201SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO. 459.8.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt 51ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG 101GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGATTCAC CTTCAGTAGC 151TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT 201GGCAGTTATA TGGTATGATG GAAGTAATGA ATACTATGCA GACTCCGTGA 251AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG 301CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG 351GGGGGCGTAC CACTTTGCCT ACTGGGGCCA GGGAACCCTG GTCACCGTCT 401CCTCAGCTTC CACCAAGGGC CCATCCGTCT TCCCCCTGGC GCCCTGCTCC 451AGGAGCACCT CCGAGAGCAC AGCCGCCCTG GGCTGCCTGG TCAAGGACTA 501CTTCCCCGAA CCGGTGACGG TGTCGTGGAA CTCAGGCGCC CTGACCAGCG 551GCGTGCACAC CTTCCCGGCT GTCCTACAGT CCTCAGGACT CTACTCCCTC 601AGCAGCGTGG TGACCGTGCC CTCCAGCAGC TTGGGCACGA AGACCTACAC 651CTGCAACGTA GATCACAAGC CCAGCAACAC CAAGGTGGAC AAGAGAGTTG 701AGTCCAAATA TGGTCCCCCA TGCCCATCAT GCCCAGCACC TGAGTTCCTG 751GGGGGACCAT CAGTCTTCCT GTTCCCCCCA AAACCCAAGG ACACTCTCAT 801GATCTCCCGG ACCCCTGAGG TCACGTGCGT GGTGGTGGAC GTGAGCCAGG 851AAGACCCCGA GGTCCAGTTC AACTGGTACG TGGATGGCGT GGAGGTGCAT 901AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TTCAACAGCA CGTACCGTGT 951GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAC GGCAAGGAGT 1001ACAAGTGCAA GGTCTCCAAC AAAGGCCTCC CGTCCTCCAT CGAGAAAACC 1051ATCTCCAAAG CCAAAGGGCA GCCCCGAGAG CCACAGGTGT ACACCCTGCC 1101CCCATCCCAG GAGGAGATGA CCAAGAACCA GGTCAGCCTG ACCTGCCTGG 1151TCAAAGGCTT CTACCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG 1201CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG 1251CTCCTTCTTC CTCTACAGCA GGCTAACCGT GGACAAGAGC AGGTGGCAGG 1301AGGGGAATGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC 1351TACACACAGA AGAGCCTCTC CCTGTCTCTG GGTAAATGA SEQ ID NO. 469.8.2 Predicted Heavy Chain Chain Protein Sequence 1mefglswvflv allrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS 51YGMHWVRQAP GKGLEWVAVI WYDGSNEYYA DSVKGRFTIS RDNSKNTLYL 101QMNSLRAEDT AVYYCARGAY HFAYWGQGTL VTVSSASTKG PSVFPLAPCS 151RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL 201SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPSCPAPEFL 251GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH 301NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT 351ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG 401QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH 451 YTQKSLSLSL GKSEQ ID NO. 47 9.8.2 Kappa Light Chain Nucleotide Sequence 1atggacatga gggtccctgc tcagctcctg gggctcctgc tgctctggct 51ctcagtcgca ggtgccagat gtGACATCCA GATGACCCAG TCTCCATCCT 101CCCTGTCTGC ATCTGTAGGA GACAGAGTCA CCATCACTTG CCAGGCGAGT 151CAGGACATTA GCAACTATTT AAATTGGTAT CAGCAGAAAC CAGGGAAAGC 201CCCTAAGCTC CTGATCTACG ATGCATCCAA TTTGGAAACA GGGGTCCCAT 251CAAGGTTCAG TGGAAGTGGA TCTGGGACAG ATTTTACTTT CACCATCAGC 301AGCCTGCAGC CTGAAGATAT TGCAACATAT TCCTGTCAAC ACTCTGATAA 351TCTCTCGATC ACCTTCGGCC AGGGGACACG ACTGGAGATT AAACGAACTG 401TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA 451TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ACCCCAGAGA 501GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC 551AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC 601AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC 651CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA 701ACAGGGGAGA GTGTTAGTGA SEQ ID NO. 489.8.2 Predicted Kappa Light Chain Protein Sequence 1mdmrvpaqll gllllwlsva garcDIQMTQ SPSSLSASVG DRVTITCQAS 51QDISNYLNWY QQKPGKAPKL LIYDASNLET GVPSRFSGSG SGTDFTFTIS 101SLQPEDIATY SCQHSDNLSI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK 151SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 201STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC SEQ ID NO. 49Nucleotide Sequence ofcynomolgus MAdCAM α₄β₇ binding domain 1ATGGATCGGG GCCTGGCCCT CCTGCTGGCG GGGCTTCTGG GGCTCCTCCA 51GCCGGGCTGC GGCCAGTCCC TCCAGGTGAA GCCCCTGCAG GTGGAGCCCC 101CGGAGCCGGT GGTGGCCGTG GCCCTGGGCG CCTCTCGCCA GCTCACCTGC 151CGCCTGGACT GCGCGGACGG CGGGGCCACG GTGCAGTGGC GGGGCCTGGA 201CACCAGCCTG GGCGCGGTGC AGTCGGACGC GGGCCGCAGC GTCCTCACCG 251TGCGCAACGC CTCGCTGTCG GCGGCCGGGA CCCGTGTGTG CGTGGGCTCC 301TGCGGGGGCC GCACCTTCCA GCACACCGTG CGGCTCCTTG TGTACGCCTT 351CCCGGACCAG CTGACCATCT CCCCGGCAGC CCTGGTGCCT GGTGACCCGG 401AGGTGGCCTG TACGGCTCAC AAAGTCACGC CTGTGGACCC CAATGCGCTC 451TCCTTCTCCC TGCTCCTGGG GGACCAGGAA CTGGAGGGGG CCCAGGCTCT 501GGGCCCGGAG GTGGAGGAGG AGGAGGAGCC CCAGGAGGAG GAGGACGTGC 551TGTTCAGGGT GACAGAGCGC TGGCGGCTGC CGACCCTGGC AACCCCTGTC 601CTGCCCGCGC TCTACTGCCA GGCCACGATG AGGCTGCCTG GCTTGGAGCT 651CAGCCACCGC CAGGCCATCC CGGTCCTGCA C SEQ ID NO. 50Amino acid sequence of cynomolgus MAdCAM α₄β₇ binding domain 1MDRGLALLLA GLLGLLQPGC GQSLQVKPLQ VEPPEPVVAV ALGASRQLTC 51RLDCADGGAT VQWRGLDTSL GAVQSDAGRS VLTVRNASLS AAGTRVCVGS 101CGGRTFQHTV RLLVYAFPDQ LTISPAALVP GDPEVACTAH KVTPVDPNAL 151SFSLLLGDQE LEGAQALGPE VEEEEEPQEE EDVLFRVTER WRLPTLATPV 201LPALYCQATM RLPGLELSHR QAIPVLH SEQ ID NO. 51Modified 6.22.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt 51ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG 101GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGATTCAC CTTCAGTAGC 151GATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT 201GGCAATTATA TGGTATGATG GAAGTAATAA ATATTATGCA GACTCCGTGA 251AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG 301CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTATATT ACTGTGCGAG 351AGATCCCGGC TACTATTACG GTATGGACGT CTGGGGCCAA GGGACCACGG 401TCACCGTCTC CTCAGCTTCC ACCAAGGGCC CATCCGTCTT CCCCCTGGCG 451CCCTGCTCTA GAAGCACCTC CGAGAGCACA GCGGCCCTGG GCTGCCTGGT 501CAAGGACTAC TTCCCCGAAC CGGTGACGGT GTCGTGGAAC TCAGGCGCTC 551TGACCAGCGG CGTGCACACC TTCCCAGCTG TCCTACAGTC CTCAGGACTC 601TACTCCCTCA GCAGCGTGGT GACCGTGCCC TCCAGCAACT TCGGCACCCA 651GACCTACACC TGCAACGTAG ATCACAAGCC CAGCAACACC AAGGTGGACA 701AGACAGTTGA GCGCAAATGT TGTGTCGAGT GCCCACCGTG CCCAGCACCA 751CCTGTGGCAG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC 801CCTCATGATC TCCCGGACCC CTGAGGTCAC GTGCGTGGTG GTGGACGTGA 851GCCACGAAGA CCCCGAGGTC CAGTTCAACT GGTACGTGGA CGGCGTGGAG 901GTGCATAATG CCAAGACAAA GCCACGGGAG GAGCAGTTCA ACAGCACGTT 951CCGTGTGGTC AGCGTCCTCA CCGTTGTGCA CCAGGACTGG CTGAACGGCA 1001AGGAGTACAA GTGCAAGGTC TCCAACAAAG GCCTCCCAGC CCCCATCGAG 1051AAAACCATCT CCAAAACCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC 1101CCTGCCCCCA TCCCGGGAGG AGATGACCAA GAACCAGGTC AGCCTGACCT 1151GCCTGGTCAA AGGCTTCTAC CCCAGCGACA TCGCCGTGGA GTGGGAGAGC 1201AATGGGCAGC CGGAGAACAA CTACAAGACC ACACCTCCCA TGCTGGACTC 1251CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC AAGAGCAGGT 1301GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC 1351AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AATGATAG SEQ ID NO. 52Modified 6.22.2 Heavy Chain Amino Acid Sequence 1mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS 51DGMHWVRQAP GKGLEWVAII WYDGSNKYYA DSVKGRFTIS RDNSKNTLYL 101QMNSLRAEDT AVYYCARDPG YYYGMDVWGQ GTTVTVSSAS TKGPSVFPLA 151PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 201YSLSSVVTVP SSNFGTQTYT CNVDHKPSNT KVDKTVERKC CVECPPCPAP 251PVAGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV QFNWYVDGVE 301VHNAKTKPRE EQFNSTFRVV SVLTVVHQDW LNGKEYKCKV SNKGLPAPIE 351KTISKTKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES 401NGQPENNYKT TPPMLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH 451NHYTQKSLSL SPGK SEQ ID NO. 53Modified 6.22.2 Kappa Light Chain Nucleotide Sequence 1atgttgccat cacaactcat tgggtttctg ctgctctggg ttccagcttc 51caggggtGAA ATTGTGCTGA CTCAGTCTCC AGACTTTCAG TCTGTGACTC 101CAAAAGAGAA AGTCACCATC ACCTGCCGGG CCAGTCAGAG AATTGGTAGT 151AGCTTACACT GGTACCAGCA GAAACCAGAT CAGTCTCCAA AACTCCTCAT 201CAAGTATGCT TCCCAGTCCT TCTCAGGGGT CCCCTCGAGG TTCAGTGGCA 251GTGGATCTGG GACAGATTTC ACCCTCACCA TCAATAGCCT GGAAGCTGAA 301GATGCTGCAA CTTATTACTG TCATCAGAGT GGTCGTTTAC CGCTCACTTT 351CGGCGGAGGG ACCAAGGTGG AGATCAAACG AACTGTGGCT GCACCATCTG 401TCTTCATCTT CCCGCCATCT GATGAGCAGT TGAAATCTGG AACTGCCTCT 451GTTGTGTGCC TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG 501GAAGGTGGAT AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG 551AGCAGGACAG CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG 601AGCAAAGCAG ACTACGAGAA ACACAAAGTC TACGCCTGCG AAGTCACCCA 651TCAGGGCCTG AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGTT 701 AGTGASEQ ID NO. 54 Modified 6.22.2 Kappa Light Chain Amino Acid Sequence 1mlpsqligfl llwvpasrgE IVLTQSPDFQ SVTPKEKVTI TCRASQRIGS 51SLHWYQQKPD QSPKLLIKYA SQSFSGVPSR FSGSGSGTDF TLTINSLEAE 101DAATYYCHQS GRLPLTFGGG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS 151VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 201SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC SEQ ID NO. 55Modified 6.34.2 Heavy Chain Nucleotide Sequence 1atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt 51ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG 101GGAGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC 151TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT 201GGCAGTTATA TCAAATGATG GAAATAATAA ATACTATGCA GACTCCGTGA 251AGGGCCGATT CACCATCTCC AGAGACAATT CCAAAAACAC GCTGTATCTG 301CAAATGAACA GCCTGCGCGC TGAGGACACG GCTGTGTATT ACTGTGCGAG 351AGATAGTACG GCGATAACCT ACTACTACTA CGGAATGGAC GTCTGGGGCC 401AAGGGACCAC GGTCACCGTC TCCTCAGCTT CCACCAAGGG CCCATCCGTC 451TTCCCCCTGG CGCCCTGCTC TAGAAGCACC TCCGAGAGCA CAGCGGCCCT 501GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA 551ACTCAGGCGC TCTGACCAGC GGCGTGCACA CCTTCCCAGC TGTCCTACAG 601TCCTCAGGAC TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAA 651CTTCGGCACC CAGACCTACA CCTGCAACGT AGATCACAAG CCCAGCAACA 701CCAAGGTGGA CAAGACAGTT GAGCGCAAAT GTTGTGTCGA GTGCCCACCG 751TGCCCAGCAC CACCTGTGGC AGGACCGTCA GTCTTCCTCT TCCCCCCAAA 801ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACGTGCGTGG 851TGGTGGACGT GAGCCACGAA GACCCCGAGG TCCAGTTCAA CTGGTACGTG 901GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCACGGG AGGAGCAGTT 951CAACAGCACG TTCCGTGTGG TCAGCGTCCT CACCGTTGTG CACCAGGACT 1001GGCTGAACGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGGCCTCCCA 1051GCCCCCATCG AGAAAACCAT CTCCAAAACC AAAGGGCAGC CCCGAGAACC 1101ACAGGTGTAC ACCCTGCCCC CATCCCGGGA GGAGATGACC AAGAACCAGG 1151TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ACCCCAGCGA CATCGCCGTG 1201GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACACCTCC 1251CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG 1301ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT 1351GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG 1401 TAAATGATAGSEQ ID NO. 56 Modified 6.34.2 Heavy Chain Amino Acid Sequence 1mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS 51YGMHWVRQAP GKGLEWVAVI SNDGNNKYYA DSVKGRFTIS RDNSKNTLYL 101QMNSLRAEDT AVYYCARDST AITYYYYGMD VWGQGTTVTV SSASTKGPSV 151FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ 201SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP 251CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV 301DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP 351APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV 401EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH 451EALHNHYTQK SLSLSPGK SEQ ID NO. 57Modified 6.34.2 Kappa Light Chain Nucleotide Sequence 1atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct 51ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT 101CTGCATCTGT CGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCAGAGT 151ATTAGTAGCT ATTTAAATTG GTATCAGCAG AAACCAGGGA AAGCCCCTAA 201GCTCCTGATC TATGCTGCAT CCGGTTTGAA GCGTGGGGTC CCATCACGGT 251TCAGTGGTAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGTTCTCTG 301CAACCTGAGG ATTTTGCAAC TTACTACTGT CACCAGAGTT ACAGTCTCCC 351ATTCACTTTC GGCCCTGGGA CCAAAGTGGA TATCAAACGA ACTGTGGCTG 401CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA 451ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA 501AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA 551GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC 601CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA 651AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG 701GAGAGTGTTA GTGA SEQ ID NO. 58Modified 6.34.2 Kappa Light Chain Amino Acid Sequence 1mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASQS 51ISSYLNWYQQ KPGKAPKLLI YAASGLKRGV PSRFSGSGSG TDFTLTISSL 101QPEDFATYYC HQSYSLPFTF GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG 151TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 201LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC SEQ ID NO. 59Modified 6.67.1 Heavy Chain Nucleotide Sequence 1atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt 51cctgtccCAG GTGCAGCTGC AGGAGTCGGG CCCAGGACTG GTGAAGCCTT 101CGGAGACCCT GTCCCTCACC TGCACTGTCT CTGGTGACTC CATCAGTAGT 151AACTATTGGA GCTGGATCCG GCAGCCCGCC GGGAAGGGAC TGGAGTGGAT 201TGGGCGTATC TATACCAGTG GGGGCACCAA CTCCAACCCC TCCCTCAGGG 251GTCGAGTCAC CATGTCAGTA GACACGTCCA AGAACCAGTT CTCTCTGAAA 301CTGAGTTCTG TGACCGCCGC GGACACGGCC GTGTATTACT GTGCGAGAGA 351TCGTATTACT ATAATTCGGG GACTTATTCC ATCCTTCTTT GACTACTGGG 401GCCAGGGAAC CCTGGTCACC GTCTCCTCAG CTTCCACCAA GGGCCCATCC 451GTCTTCCCCC TGGCGCCCTG CTCTAGAAGC ACCTCCGAGA GCACAGCGGC 501CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT 551GGAACTCAGG CGCTCTGACC AGCGGCGTGC ACACCTTCCC AGCTGTCCTA 601CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG 651CAACTTCGGC ACCCAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA 701ACACCAAGGT GGACAAGACA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA 751CCGTGCCCAG CACCACCTGT GGCAGGACCG TCAGTCTTCC TCTTCCCCCC 801AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG 851TGGTGGTGGA CGTGAGCCAC GAAGACCCCG AGGTCCAGTT CAACTGGTAC 901GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCAC GGGAGGAGCA 951GTTCAACAGC ACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGG 1001ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC 1051CCAGCCCCCA TCGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA 1101ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC 1151AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC 1251TCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 1301TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 1351CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 1401GGGTAAATGA TAG SEQ ID NO. 60Modified 6.67.1 Heavy Chain Amino Acid Sequence 1mkhlwfflll vaaprwvlsQ VQLQESGPGL VKPSETLSLT CTVSGDSISS 51NYWSWIRQPA GKGLEWIGRI YTSGGTNSNP SLRGRVTMSV DTSKNQFSLK 101LSSVTAADTA VYYCARDRIT IIRGLIPSFF DYWGQGTLVT VSSASTKGPS 151VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL 201QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP 251PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY 301VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL 351PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA 401VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM 451HEALHNHYTQ KSLSLSPGK SEQ ID NO. 61Modified 6.67.1 Kappa Light Chain Nucleotide Sequence 1atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg 51tgcctacggg GACATCGTGA TGACCCAGTC TCCAGACTCC CTGGCTGTGT 101CTCTGGGCGA GAGGGCCACC ATCAACTGCA AGTCCAGCCA GAGTGTTTTA 151TACAGCTCCA ACAATAAGAA CTACTTAGCT TGGTACCAAC AGAAACCAGG 201ACAGCCTCCT AAATTGCTCA TTTACTGGGC ATCTATACGG GAATATGGGG 251TCCCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC 301ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTTTATTTCT GTCAACAATA 351TTATAGTATT CCTCCCCTCA CTTTCGGCGG AGGGACCAAG GTGGAGATCA 401AACGAACTGT GGCTGCACCA TCTGTCTTCA TCTTCCCGCC ATCTGATGAG 451CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA ATAACTTCTA 501TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG 551GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC 601AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA 651AGTCTACGCC TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA 701AGAGCTTCAA CAGGGGAGAG TGTTAGTGA SEQ ID NO. 62Modified 6.67.1 Kappa Light Chain Amino Acid Sequence 1mvlqtqvfis lllwisgayg DIVMTQSPDS LAVSLGERAT INCKSSQSVL 51YSSNNKNYLA WYQQKPGQPP KLLIYWASIR EYGVPDRFSG SGSGTDFTLT 101ISSLQAEDVA VYFCQQYYSI PPLTFGGGTK VEIKRTVAAP SVFIFPPSDE 151QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY 201SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C SEQ ID NO. 63Modified 6.77.1 Heavy Chain Nucleotide Sequence 1atggaactgg ggctccgctg ggttttcctt gttgctattt tagaaggtgt 51ccagtgtGAG GTGCAGCTGG TGGAGTCTGG GGGAGGCCTG GTCAAGCCTG 101GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC 151TATAGCATGA ACTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT 201CTCATCCATT AGTAGTAGTA GTAGTTACAT ATACTACGCA GACTCAGTGA 251AGGGCCGATT CACCATCTCC AGAGACAACG CCAAGAACTC ACTGTATCTG 301CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG 351AGATGGGTAT AGCAGTGGCT GGTCCTACTA CTACTACTAC GGTATGGACG 401TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAGCTTC CACCAAGGGC 451CCATCCGTCT TCCCCCTGGC GCCCTGCTCT AGAAGCACCT CCGAGAGCAC 501AGCGGCCCTG GGCTGCCTGG TCAAGGACTA CTTCCCCGAA CCGGTGACGG 551TGTCGTGGAA CTCAGGCGCT CTGACCAGCG GCGTGCACAC CTTCCCAGCT 601GTCCTACAGT CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACCGTGCC 651CTCCAGCAAC TTCGGCACCC AGACCTACAC CTGCAACGTA GATCACAAGC 701CCAGCAACAC CAAGGTGGAC AAGACAGTTG AGCGCAAATG TTGTGTCGAG 751TGCCCACCGT GCCCAGCACC ACCTGTGGCA GGACCGTCAG TCTTCCTCTT 801CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA 851CGTGCGTGGT GGTGGACGTG AGCCACGAAG ACCCCGAGGT CCAGTTCAAC 901TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCACGGGA 951GGAGCAGTTC AACAGCACGT TCCGTGTGGT CAGCGTCCTC ACCGTTGTGC 1001ACCAGGACTG GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA 1051GGCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAACCA AAGGGCAGCC 1101CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA 1151AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC 1201ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC 1251CACACCTCCC ATGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC 1301TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC 1351GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT 1401GTCTCCGGGT AAATGATAG SEQ ID NO. 64Modified 6.77.1 Heavy Chain Protein Sequence 1melglrwvfl vailegvqcE VQLVESGGGL VKPGGSLRLS CAASGFTFSS 51YSMNWVRQAP GKGLEWVSSI SSSSSYIYYA DSVKGRFTIS RDNAKNSLYL 101QMNSLRAEDT AVYYCARDGY SSGWSYYYYY GMDVWGQGTT VTVSSASTKG 151PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA 201VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE 251CPPCPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN 301WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK 351GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD 401IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS 451VMHEALHNHY TQKSLSLSPG K SEQ ID NO. 65Modified 6.77.1 Kappa Light Chain Nucleotide Sequence 1atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg 51atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA 101CTCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG 151CTTAGTGATG GAAAGACCTA TTTGAATTGG TACCTGCAGA AGCCCGGCCA 201GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC 251CAGACAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC 301AGCCGGGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAAGTAT 351ACAGCTTATG TGCAGTTTTG GCCAGGGGAC CAAGCTGGAG ATCAAACGAA 401CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG 451AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG 501AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT 551CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC 601AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA 651CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT 701TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO. 66Modified 6.77.1 Kappa Light Chain Amino Acid Sequence 1mrlpaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL 51LSDGKTYLNW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI 101SRVEAEDVGV YSCMQSIQLM SSFGQGTKLE IKRTVAAPSV FIFPPSDEQL 151KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 201SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC SEQ ID NO. 67Modified 7.26.4 Kappa Light Chain Nucleotide Sequence 1atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg 51atccagtgcg GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA 101CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG 151TATAGTGATG GAAAGACCTA TTTGTTTTGG TACCTGCAGA AGCCAGGCCA 201GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGATTC TCTGGAGTGC 251CAGATAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC 301AGCCGGGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAAGTAT 351ACAGCTTCCG TGGACGTTCG GCCAAGGGAC CAAGGTGGAA ATCAAACGAA 401CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG 451AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG 501AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT 551CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC 601AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA 651CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT 701TCAACAGGGG AGAGTGTTAG TGA SEQ ID NO. 68Modified 7.26.4 Kappa Light Chain Amino Acid Sequence 1mrlpaqllgl lmlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL 51YSDGKTYLFW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDTTLKI 101SRVEAEDVGV YYCMQSIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL 151KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 201SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

1. (canceled)
 2. A human monoclonal antibody or antigen-binding portionthereof that specifically binds to Mucosal Adressin Cell AdhesionMolecule (MAdCAM), wherein said antibody or portion possesses at leastone of the following properties: (a) binds to human cells; (b) has aselectivity for MAdCAM over VCAM or fibronectin of at least 100 fold;(c) binds to human MAdCAM with a K_(d) of 3×10⁻¹⁰ M or less; or (d)inhibits the binding of α₄β₇ expressing cells to human MAdCAM. (e)inhibits the recruitment of lymphocytes to gastrointestinal lymphoidtissue.
 3. (canceled)
 4. A hybridoma cell line that produces the humanmonoclonal antibody according to claim claim 2, wherein the hybridoma isselected from the group consisting of 1.7.2 (ECACC Accession No.03090901), 1.8.2 (ECACC Accession No. 03090902), 6.14.2 (ECACC AccessionNo. 03090903), 6.22.2 (ECACC Accession No. 03090904), 6.34.2 (ECACCAccession No. 03090905), 6.67.1 (ECACC Accession No. 03090906), 6.73.2(ECACC Accession No. 03090907), 6.77.1 (ECACC Accession No. 03090908),7.16.6 (ECACC Accession No. 03090909), 7.20.5 (ECACC Accession No.03090910), 7.26.4 (ECACC Accession No. 03090911), and 9.8.2 (ECACCAccession No. 03090912).
 5. The human monoclonal antibody produced bythe hybridoma cell line according to claim 4 or an antigen-bindingportion of said monoclonal antibody.
 6. (canceled)
 7. The humanmonoclonal antibody or antigen-binding portion thereof according toclaim 1, wherein said antibody or antigen-binding portion inhibitsbinding of human MAdCAM to α₄β₇, and wherein the antibody or portionthereof has at least one of the following properties: (a) cross-competeswith a reference antibody for binding to MAdCAM; (b) competes with areference antibody for binding to MAdCAM; (c) binds to the same epitopeof MAdCAM as a reference antibody; (d) binds to MAdCAM withsubstantially the same K_(d) as a reference antibody; (e) binds toMAdCAM with substantially the same off rate as a reference antibody;wherein the reference antibody is selected from the group consisting of:monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonalantibody 6.14.2, monoclonal antibody 6.22.2, monoclonal antibody 6.34.2,monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonalantibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5,monoclonal antibody 7.26.4, monoclonal antibody 9.8.2, monoclonalantibody 6.22.2-mod, monoclonal antibody 6.34.2-mod, monoclonal antibody6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody7.26.4-mod.
 8. The monoclonal antibody of claim 5, wherein the antibodyis selected from the group consisting of: (a) an antibody comprising theamino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4, withoutthe signal sequences; (b) an antibody comprising the amino acidsequences set forth in SEQ ID NO: 6 and SEQ ID NO: 8, without the signalsequences; (c) an antibody comprising the amino acid sequences set forthin SEQ ID NO: 10 and SEQ ID NO: 12, without the signal sequences; (d) anantibody comprising the amino acid sequences set forth in SEQ ID NO: 14and SEQ ID NO: 16, without the signal sequences; (e) an antibodycomprising the amino acid sequences set forth in SEQ ID NO: 18 and SEQID NO: 20, without the signal sequences; (f) an antibody comprising theamino acid sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 24,without the signal sequences; (g) an antibody comprising the amino acidsequences set forth in SEQ ID NO: 26 and SEQ ID NO: 28, without thesignal sequences; (h) an antibody comprising the amino acid sequencesset forth in SEQ ID NO: 30 and SEQ ID NO: 32, without the signalsequences; (i) an antibody comprising the amino acid sequences set forthin SEQ ID NO: 34 and SEQ ID NO: 36, without the signal sequences; (j) anantibody comprising the amino acid sequences set forth in SEQ ID NO: 38and SEQ ID NO: 40, without the signal sequences; (k) an antibodycomprising the amino acid sequences set forth in SEQ ID NO: 42 and SEQID NO: 44, without the signal sequences; (l) an antibody comprising theamino acid sequences set forth in SEQ ID NO: 46 and SEQ ID NO: 48,without the signal sequences; (m) an antibody comprising the amino acidsequences set forth in SEQ ID NO: 52 and SEQ ID NO: 54, without thesignal sequences; (n) an antibody comprising the amino acid sequencesset forth in SEQ ID NO: 56 and SEQ ID NO: 58, without the signalsequences; (o) an antibody comprising the amino acid sequences set forthin SEQ ID NO: 60 and SEQ ID NO: 62, without the signal sequences; (p) anantibody comprising the amino acid sequences set forth in SEQ ID NO: 64and SEQ ID NO: 66, without the signal sequences; and (q) an antibodycomprising the amino acid sequences set forth in SEQ ID NO: 42 and SEQID NO: 68, without the signal sequences.
 9. A monoclonal antibody or anantigen-binding portion thereof, wherein the heavy chain of saidantibody or portion thereof comprises the heavy chain CDR1, CDR2 andCDR3 or wherein the light chain comprises the light chain CDR1, CDR2 andCDR3 of a monoclonal antibody selected from the group consisting of:1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6,7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-modand 7.26.4-mod. 10-11. (canceled)
 12. The human monoclonal antibody orantigen-binding portion according to claim 2, wherein the heavy chainvariable region, the light chain variable region or both are at least90% identical in amino acid sequence to the corresponding region orregions of a monoclonal antibody selected from the group consisting of:monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonalantibody 6.14.2, monoclonal antibody 6.22.2, monoclonal antibody 6.34.2,monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonalantibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5,monoclonal antibody 7.26.4 monoclonal antibody 9.8.2, monoclonalantibody 6.22.2-mod, monoclonal antibody 6.34.2-mod, monoclonal antibody6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody7.26.4-mod.
 13. A monoclonal antibody or an antigen-binding portionthereof that specifically binds MAdCAM according to claim 2, wherein:(a) the heavy chain comprises the heavy chain CDR1, CDR2 and CDR3 aminoacid sequences of a reference antibody selected from the groupconsisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod and 7.26.4-mod (b) the light chain comprises thelight chain CDR1, CDR2 and CDR3 amino acid sequences of a referenceantibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod (c) theantibody comprises a heavy chain of (a) and a light chain of (b); and(d) the antibody of (c) wherein the heavy chain and light chain CDRamino acid sequences are selected from the same reference antibody.14.-17. (canceled)
 18. A pharmaceutical composition comprising aneffective amount of the monoclonal antibody or antigen-binding portionthereof according to claim 2 and a pharmaceutically acceptable carrier.19. A method of treating inflammatory disease in a subject in needthereof, comprising the step of administering to said subject themonoclonal antibody or antigen-binding portion thereof according toclaim 2 wherein said antibody or antigen-binding portion inhibitsbinding of MAdCAM to α₄β₇. 20.-24. (canceled)
 25. The cell lineaccording to claim 2 that produces an antibody selected from the groupconsisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2, or an antibody comprising theamino acid sequences of one of said antibodies.
 26. (canceled)
 27. Anisolated nucleic acid molecule comprising a nucleotide sequence thatencodes the heavy chain or an antigen-binding portion thereof or thelight chain or an antigen-binding portion thereof of an antibodyaccording to claim
 2. 28.-33. (canceled)
 34. A method of treating asubject in need thereof with a human antibody or antigen-binding portionthereof that specifically binds to MAdCAM and inhibits binding to α₄ β₇comprising the steps of: (a) administering an effective amount of anisolated nucleic acid molecule encoding the heavy chain or anantigen-binding portion thereof, an isolated nucleic acid moleculeencoding the light chain or an antigen-binding portion thereof, ornucleic acid molecules encoding the light chain and the heavy chain orantigen-binding portions thereof; and (b) expressing the nucleic acidmolecule.
 35. A method for producing a human monoclonal antibody thatspecifically binds MAdCAM, comprising the steps of: (a) immunizing anon-human transgenic animal that is capable of producing humanantibodies with MAdCAM, with an immunogenic portion of MAdCAM or a withcell or tissue expressing MAdCAM; and (b) allowing the transgenic animalto mount an immune response to MAdCAM. 36.-41. (canceled)
 42. Amonoclonal antibody or an antigen-binding portion thereof of claim 5that specifically binds MAdCAM, and wherein said antibody or portionthereof comprises one or more of an FR1, FR2, FR3 or FR4 amino acidsequence of a human monoclonal antibody selected from the groupconsisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2,6.77.1, 7.16.6, 7.20.5, 7.26.4 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod and 7.26.4-mod.
 43. The human monoclonal antibodyor antigen-binding portion according to claim 2, wherein the antibodycomprises: (a) a heavy chain amino acid sequence that is at least 90%identical to the heavy chain amino acid sequence of a monoclonalantibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2,6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2,6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod; (b) alight chain amino acid sequence that is at least 90% identical to thelight chain amino acid sequence of a monoclonal antibody selected fromthe group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1,6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod,6.67.1-mod, 6.77.1-mod and 7.26.4-mod; (c) both (a) and (b); or (d)either (a), (b) or (c), with or without the signal sequence.
 44. Amethod for diagnosing a disorder characterized by circulating solublehuman MAdCAM comprising the steps of: (1) contacting a biological samplewith the monoclonal antibody according to claim 2 or antigen-bindingportion and (2) detecting binding.
 45. A method for detectinginflammation in a subject comprising the steps of: (1) administering tosaid subject the monoclonal antibody or antigen-binding portionaccording to claim 2 wherein said antibody or portion thereof isdetectably labeled and (2) detecting binding. 46.-48. (canceled)
 49. Avaccine comprising an effective amount of the human antibody thereofaccording to claim 2 or antigen-binding portion and a pharmaceuticallyacceptable carrier.
 50. (canceled)
 51. A method of detecting the effectof administration of an inhibitory anti-MAdCAM antibody orantigen-binding portion thereof to a subject comprising the steps of:(a) administering to a subject a human monoclonal antibody thatspecifically binds to MAdCAM; and (b) determining whether there is anincrease in the levels of circulating α₄β₇-expressing leukocytes.52.-53. (canceled)